Formulations containing hyaluronic acid

ABSTRACT

Topically applied transdermally quick penetrating compositions containing a drug and hyaluronic acid and/or salts thereof and methods of treating diseases and conditions employing said compositions.

This invention is a Continuation-In-Part Application of U.S. patentapplication Ser. No. 07/675,908, first filed under PCT (PatentCooperation Treaty) Application Serial No. PCT/CA90/00306 filed on theSep. 18, 1990 (claiming priority from Canadian Patent Application SerialNo. 612,307, filed the Sep. 21, 1989) and entering the National Phase inthe United States on the Jul. 3, 1991. This invention is also aContinuation-In-Part Application of U.S. patent application Ser. No.07/838,674, filed on the Feb. 21, 1992, now abandoned. This applicationis also a Divisional Application of U.S. patent application Ser. No.08/290,840, filed on the Oct. 27, 1994.

FIELD OF INVENTION

This invention relates to the treatment of disease and conditions of theskin and exposed tissue. In some embodiments this invention findsapplication to the treatment of a disease or condition of the skin andexposed tissue including basal cell carcinoma, squamous cell tumours,metastatic cancer of the breast to the skin, primary and metastaticmelanoma in the skin, malignancies and tumours in the skin, genitalwarts (condyloma acuminata), cervical cancer, HPV (Human PapillomaVirus) including HPV (Human Papilloma Virus) on the cervix, psoriasis(both plaque-type psoriasis and nail bed psoriasis), corns on the feet,actinic keratoses lesions, "liver" spots, fungal lesions, and other suchtypes of lesions, and hair loss on the head of a pregnant women.

This invention also relates to compositions and formulations suitablefor use in such treatments, the use of such formulations in suchtreatments, methods of such treatment, and the delivery of drugs forsuch treatments.

BACKGROUND OF THE INVENTION

Basal cell carcinoma is presently treated by surgery. Each lesion,together with all surrounding and underlying tissue (dermis, epidermis,and subdermis), is cut out. In some instances, surgery, while necessaryfor the patient's welfare, puts the patient at risk in some otherrespect (for example, the removal of a lesion on a patient's temple(resection) may jeopardize the patient's health). Squamous cell tumoursare also treated the same way as are other forms of cancer in the skinand exposed tissue. Furthermore, other conditions and diseases of theskin and exposed tissue are treated in the same way or in ways thatcause discomfort to the patient, for example melanoma, genital warts,cervical cancer, HPV (Human Papilloma Virus).

Actinic keratoses lesion is dealt with similarly. Liquid nitrogen isalso used to remove the lesion.

These diseases and conditions are usually found in the epidermis (atleast for the most part, extending into the dermis and upwards throughthe Stratum Corneum).

Hyaluronic acid is a naturally occurring glycosaminoglycan. Itsmolecular weight may vary from 50,000 daltons upwards, and it formshighly viscous solutions. As regards the actual molecular weight ofhyaluronic acid in natural biological contexts, this is still a matterof much uncertainty; when the molecular weight of hyaluronic acid is tobe determined, different values are obtained depending on the assaymethod employed, and on the source, the isolation method etc. The acidoccurs in animal tissue, e.g. spinal fluid, ocular fluid, synovialfluid, cockscombs, skin, and also in some streptococci. Various gradesof hyaluronic acid have been obtained. A preparation with an allegedlyhigh degree of purity and alleged to be entirely free from side effects,is a non-inflammatory form described in U.S. Pat. No. 4,141,973; thispreparation is said to have a molecular weight exceeding 750,000 dalton,preferably exceeding 1,200,000 dalton and is suggested for therapeuticuse in various articular conditions. Applicants believe that hyaluronicacid claimed in this patent is sold under the trade mark Healon.

U.S. Pat. No. 4,801,619 relates to hyaluronic acid, having a molecularweight of about 3×10⁶ dalton or more, administered intra-articularlywhich is prone to decrease the proteoglycan content of synovial fluid toalmost normal levels. According to this patent, this indicates apositive effect on the proteoglycan metabolism of a joint. According tothe patent, this is applicable both to inflammatory conditions and todegeneration caused by treatment with symptomatics, such ascorticosteroid preparations. It is thus clear that a sufficiently highmolecular weight of the hyaluronic acid is alleged to counteract sideeffects that might be caused by corticosteroids or other symptomaticsproducing similar effects. When corticosteroids are applied, the amountof hyaluronic acid in the synovial cavity will, according to the patent,increase substantially and, according to the inventors, their hyaluronicacid preparations have a very positive effect on such clinical symptomsas pain, swelling, and lameness.

The patent states that the objectives of the invention are attained byintra-articular administration (injection) of an effective amount ofhyaluronic acid with a mean molecular weight exceeding 3×10⁶ dalton,preferably exceeding 4×10⁶ dalton; usually the molecular weight will notexceed 7×10⁶ dalton. The dosage of hyaluronic acid administered isstated to be preferably within the range of 5 mg-80 mg. The amount ofsolution given at each administration is generally less than 60 ml, e.g.less than 20 ml. of an aqueous solution of the acid or its salt. It isconvenient to administer the acid dissolved in water (<2% w/w, bufferedto physiological pH), for instance in the form of a water-soluble sodiumsalt. The exact amount will depend on the particular joint to betreated.

The Merck Index Specifies that Hyaluronic Acid has a Molecular Weightwithin the range pf 50,000 to 8×10⁶ depending on source, methods ofpreparation, and methods of determination. The Merck Publication teacheshyaluronic acid as a surgical aid (ophthalmological).

U.S. Pat. No. 4,808,576 purports to teach that hyaluronic acid, an agentwell known for reducing the sequelae of trauma in mammalian joint tissuewhen injected directly into the traumatized joint tissue, will becarried to such traumatized tissue by the mammal's natural processes ifapplied at a site remote from the traumatized tissue. Thus, hyaluronicacid in any therapeutically acceptable form can, according to thePatent, be administered by the typical remote routes includingintravenous, intramuscular, subcutaneous, and topical.

This, the patent alleges, makes the utilization of hyaluronic acid muchmore convenient and attractive. For instance, the treatment of arthritisin horse or human joints with hyaluronic acid, according to the patent,no longer requires more difficult intra-articular injections.

U.S. Pat. No. 4,725,585 relates to a method of enhancing or regulatingthe host defence of a mammal by administering to a mammal atherapeutically effective amount of hyaluronic acid.

At column 1, lines 43-46, the patent provides that he invention wasbased on the unexpected discovery that administration of hyaluronic acidto mammals results in a considerable increase in the defence.

The hyaluronic acid employed in the patent was Healon (t.m) provided byPharmacia AB, Uppsala, Sweden (Pharmacia AB is also entitled to thebenefit of U.S. Pat. No. 4,141,973). The patent provides at column 4,line 19 that because a patient's infections had been hard to treat,instead of just hyaluronic acid being administered to the patient toincrease the patient's defence, the patient was given hyaluronic acidand an antibiotic. While one reading the patent may conclude that theantibiotic was given in combination with hyaluronic acid, in factbecause the hyaluronic acid was administered subcutaneously and becausethe patient was a heart patient, one skilled in the art would understandthat any antibiotic administered, while possibly administeredsimultaneously with the administration of the hyaluronic acid, wasdefinitely administered separately intravenously (probably) orintramuscularly (less probably). Thus, the hyaluronic acid administered,according to the teachings of this patent, was administered in order toprevent possible development of infections (increase the host's defence)and not for any other reason.

U.S. Pat. No. 4,636,524 discloses cross-linked gels of hyaluronic acid,alone and mixed with other hydrophilic polymers and containing varioussubstances or covalently bonded low molecular weight substances andprocesses for preparing them. These products are alleged to be useful innumerous applications including cosmetic formulations and as drugdelivery systems.

The patent further states that as hyaluronic acid is known to be abiologically tolerable polymer in the sense that it does not cause anyimmune or other kind of response when introduced into a human body, thecross-linked hyaluronic acid gels can be used for various medicalapplications. The cross-linked gels modified with other polymers or lowmolecular weight substances, it is alleged, can be used as drug deliverydevices. For example, the inventors are alleged to have found thatheparin introduced in a cross-linked hyaluronic acid gel retained itsantithrombogenic activity.

The inventors also allege that they have also found that cross-linkedgels of hyaluronic acid can slow down the release of a low molecularweight substance dispersed therein but not covalently attached to thegel macromolecular matrix.

U.S. Pat. No. 4,736,024 purports to teach new medicaments for topicaluse containing:

(i) an active pharmacological substance or a mixture of pharmacologicalsubstances, either active or suitable for topical administration and

(ii) a topical vehicle which comprises hyaluronic acid or a molecularfraction of hyaluronic acid or a salt of the same with an alkalinemetal, an alkaline earth metal, magnesium, aluminium, ammonium, or apharmacological substance optionally together with additionalconventional excipients for pharmaceutical preparations for topical use.

Applicants are also aware of published Japanese Patent Document61000017, dated Jan. 6, 1986, whose English abstract of disclosurestates that the Japanese Patent Document relates to the use ofhyaluronic acid or cross-linked hyaluronic acid or their salts as theactive ingredient for inhibiting carcinoma metastasis.

According to the purported abstract of the patent, more that 1.0% ofhyaluronic acid is dissolved in alkaline aq. soln. and pref. more than50% of H₂ O sol. org. solvent. eq. alcohol, acetone, dioxane, againsttotal soln. is added. Preferably the pH is 12-14. Then a multifunctionalepoxy cpd. is added and reacted at 10-60 deg. C., pref. at 20-40-deg. C.for 24 hrs. Cross-linking ratio of crosslinked hyaluronic acid or itssalt is regulated by changing mol ratio of hyaluronic acid or its saltand multifunctional epoxy cpd.. Pref. hyaluronic acid used has intrinsicviscosity 0.2-30,m.w. 4000-2000000. The hyaluronic acid is allegedlyused in several dosage forms. The clinical dose for an adult is allegedto be normally, as hyaluronic acid or cross-linked hyaluronic acid, 25mg-5 g/day (p.o.) and 10 mg-2.5 g/l dose (inj). The abstract allegesthat the advantage is that the hyaluronic acid has no side effects asmay other anti-cancer drugs and has an analgesic and a tissuerestoration effect.

European Patent Application 0295092 purports to teach a vehicle togetherwith fragments of hyaluronic acid for delivering of the fragments ofhyaluronic acid into the skin to reach the dermal layer of the skin toincrease the development of blood vessels for stimulating hair growth orregrowth. The preferred fragments of hyaluronic acid are polysaccharidescontaining from 7 to 25 monosaccharide units. The patent provides thatit is apparent that the larger the fragments of hyaluronic acid, thegreater the difficulty there is in delivering the fragments to thedermal layer of the skin, unless there is also present in thecomposition a means for enhancing the activity of said fragments.

The combination may thus include a means for enhancing the activity ofthe fragments of hyaluronic acid, especially to improve theirpenetration through the skin following topical application. Someactivity enhancers, it is alleged, also function as vehicles for thefragments of the hyaluronic acid.

Some activity enhancers are also alleged to possess the ability tostimulate or increase hair growth. Minoxidil is asserted among others tobe such an activity enhancer. Thus both the fragments of hyaluronic acidand minoxidil are alleged to stimulate hair growth both delivered by avehicle.

European Patent Application 0179442 asserts that where free radicals areformed in considerable quantities, hyaluronic acid is broken down ordegraded before the hyaluronic acid has given the desired effect.

Canadian Letters Patent 1,240,929 teaches the combination of chondroitinsulfate compound and a hyaluronate to protect both human and animal celllayers and tissue subject to exposure to trauma.

European Patent Application 0208623 purports to teach hyaluronic acid as"une augmentation de l'activite de certaines proteases". It alsopurports to teach the use of hyaluronic acid for treating connectivetissue diseases, including malignant tumours and cardiovasculardisorders.

European Patent Application 270317 purports to teach the combination ofan antiviral agent lacking inhibitory action and a compound for example,hyaluronic acid! possessing cell fusion inhibitory activity and/orvirus-adsorption inhibitory activity for treating disease carried by avirus.

U.S. Pat. No. 4,840,941 purports to teach the use of an effective amountof hyaluronic acid as the active agent for the treatment of retrovirusesin association with a pharmaceutically acceptable carrier, diluent, orexcipient.

U.S. Pat. No. 4,851,521 and European Patent Application 0265116 bothdescribe hyaluronic acid fractions, the making thereof and cross-linkedesters of hyaluronic. U.S. Pat. No. 4,851,521 describes esters ofhyaluronic acid incorporated into pharmaceutical preparations as theactive ingredient and as vehicles for ophthamological medicines fortopical use (See column 11, lines 35 to 42; and column 12, lines 62 tocolumn 13, line 3) and in suppositories for a systemic effect due to theeffect of transcutaneous absorption, such as in suppositories.

The patent provides at column 13, lines 5 to 31: "The vehicling actionof the hyaluronic esters also applies to associated medicaments of thetype mentioned above in which the active substance acts not onlytopically or by nasal or rectal absorption, for example by nasal spraysor preparations for inhalation for the oral cavity or the pharynx, butalso by oral or parenteral route, for example by intramuscular,subcutaneous or intravenous route, as it favors absorption of the druginto the application site. The new medicaments can therefore be applied,apart from in the fields already mentioned, in practically all sectorsof medicine, such as internal medicine, for example in pathologies ofthe cardiovascular system, in infections of the respiratory system, thedigestive system, the renal system, in diseases of an endocrinologicalnature, in oncology, in psychiatry etc., and may also be classifiedtherefore from the point of view of their specific action, being perhapsanesthetics, analgesics, anti-inflammatories, wound healers,antimicrobics, adrenergic agonists and antagonists, cytostatics,antirheumatics, antihypertensives, diuretics, sexual hormones,immunostimulants and immunosuppressants, for example, one of the drugshaving the activity already described for the therapeutically activealcohols to be used as esterifying component according to the presentinvention, or for the therapeutically active bases used for thesalification of the free carboxylic groups."

There have been extensive studies to determine the defect in immunefunction that allows a tumour cell to develop. It was postulatedinitially by Jerne, and subsequently by Burnett, that the immunesystem's major role was that of immunological surveillance to destroyabnormal cells. The concept of surveillance, while somewhat simplistic,remains an accepted concept for the elaborate mechanism of immunerecognition and function that is present in the higher species--mammals.

It has then been postulated that tumours develop because of local orgeneralized immune suppression. However, as pointed out by Moller, ifgeneral immune suppression occurs, it is only certain types ofneoplastic disorders that develop, mainly those of the lympho-reticularsystem. This observation is generally correct and represents a majorchallenge to the immune surveillance theory unless a specific reason canbe shown as to why the individual cancer cell can develop plusindividually evade the immune system.

It was demonstrated experimentally in 1974 that defects of macrophagefunction may exist in neoplastic disease.

The initial experiments found suppressor cells to be part of the immunesystem; these were either of the T-cell type of the macrophage cellsystem. There was presence demonstrated in neoplasia, chronic bacterialinfection, recovery from massive injury and chronic fungal infection.

There has been repeated demonstration in experimental animals that themacrophage cell function is altered in neoplastic disease. Themacrophages in the animal's systems appeared "blocked" in theirfunction. Generally when removed from the in vivo situation, washed insaline and cultured, they perform normally. This block has been shown tobe related to the excessive production of prostaglandin by neoplastictissue or by the macrophage itself. Similarly, the N.K. cells (which aresaid to be primitive or immature macrophages and which may be involvedin cancer defence) are also blocked.

In the basic research efforts in the latter '70s and the early '80's,there existed considerable confusion as to what role immunotherapyshould take in cancer. Activation or "hyping" of macrophages was thoughtto be important. However, in an examination by Romans and Falk ofperitoneal macrophages obtained from patients with neoplastic disease,there was definite evidence that these macrophages were alreadyactivated yet were co-existing with cancer cells and not causing theirdestruction.

It has recently been shown by several independent investigators that themalfunction of macrophages or the putitive block is due to excessiveprostaglandin and that this can be altered in tissue culture bycorticosteroids, ASA, and the non-steroidal anti-inflammatory drugs,i.e. indomethacin and naproxen (Naprosyn™). Again, it was repeatedlydemonstrated that in animal tumours these substances could alter theresponse to neoplastic cells and that various combinations of thesesubstances employed with immune enhancing agents could produce verycredible success in eliminating experimental tumours. Lala andco-workers combined Indomethacin therapy with Interleukin 2 and showedthat this could effect a cure with experiment neoplasm.

There were continued problems with the use of any of these agents in theactual human in vivo experience. All of the non-steroidalanti-inflammatory agents (NSAID) produced major toxicity in terms ofgastro-intestinal, neurological, and other areas. Thus, the basis of thepresent approach is that, under general circumstances, with the use ofthese agents in human disease in sufficient amounts, the drug willpenetrate to any pathological tissue to alter therapeutically localprostaglandin production. While intravenous preparations of Indomethacin(and now of other agents) exist, using these drugs alone producesprohibitive side effects in human subjects. Therefore, only insufficientamounts can be brought into the body to effect more than occasionalresponses in neoplasm.

However, the majority of the evidence is present to indicate andtherefore, it can be postulated that the basis for neoplasticdevelopment and how the initial cell "sneaks by" the immune surveillancemechanism relates to its production of prostaglandin. One need postulateonly one mutation to alter the amount of prostaglandin synthesisproduced by cells when they become "malignant" to establish a mechanismof blocking out the initial cell in any immune reaction, i.e. themacrophage. It therefore became essential to develop a combination ofNSAIDs for clinical use to produce a major improvement in response inneoplastic disease and other conditions where excessive prostaglandinsynthesis represents the basis of the pathogenesis of this diseasestate, i.e. arthritis and various others of the so-called connectivetissue inflammatory disorders and/or auto-aggressive diseases.

See also:

1. Modulation of Immunity in Cancer Patients by ProstaglandinAntagonists, Immunity to Cancer II, Alan R. Liss, Inc.; and

2. Goodwin, J. S., (1981) Prostaglandin E and Cancer Growth Potentialfor Immunotherapy with Prostaglandin Synthesis Inhibitors, AugmentiveAgents in Cancer Therapy, Raven Press, New York.

U.S. Pat. No. 4,711,780 teaches a pharmaceutical composition comprisingVitamin C, a zinc salt, and a sulfur amino acid for treating surfaceepithelium for epithelium regeneration. Hyaluronic acid may be added forapplications in the reproductive tract to block the passage of toxinsinto the blood system.

U.S. Pat. No. 4,937,254 (Ethicon) teaches combinations of hyaluronicacid and salts thereof with NSAIDS for the prevention of adhesions aftersurgery.

Because of the side effects of the use of non-steroidalanti-inflammatory drugs (major toxicity in terms of gastro-intestinal,neurological, and other areas), use thereof should also be restricted(if possible) to the area of use without delivery to other areas whichare not in need of treatment. Thus, if useful amounts of thenon-steroidal anti-inflammatory drugs or for that matter any drugs couldbe delivered to a site in need thereof without carriage of substantialamounts away from the site to be treated, thereby accumulating an amountof the drug at the site to be treated for a prolonged period of time,then the use of the drug for example a non-steroidal anti-inflammatorydrug at a site may have many other useful applications.

SUMMARY OF THE INVENTION

Applicants have now developed compositions, (combinations andformulations) which are topically applied to the skin and/or exposedtissue of a human and which are quickly transported in dosage amountspercutaneously (intracutaneously) at a site in need of treatment, (siteof pathology and/or trauma) best targeting the epidermis andsubsequently remaining (accumulating) at the site for a prolonged periodof time. The compositions subsequently clear through the lymphaticsthereby bringing dosage amounts of the compositions to the lymphaticsfor the treatment of disease and conditions in the lymphatics.

These compositions, (combinations and formulations) employ, combine, orincorporate (as the case may be) a plurality of effective non-toxicdosage amounts, each dosage amount comprising an effective non-toxicdosage amount of a drug for example a drug which inhibits prostaglandinsynthesis for example an NSAID and an effective non-toxic dosage amountof a form of hyaluronic acid (preferably hyaluronic acid or saltthereof) for the transport of the drug to the site of the pathologyand/or trauma. Suitable dosage amounts of the composition may be removedfrom a container (for example a tube or jar) and administered (forexample, applied).

Thus according to one aspect of the invention these pharmaceuticalcompositions (combinations and formulations) comprise a plurality ofeffective non-toxic dosage amounts for administration to the skin and/orexposed tissue of a human in need of treatment, each such dosage amountcomprising a therapeutically effective non-toxic (to the patient) dosageamount of a drug to treat a disease and/or condition for example a drugwhich inhibits prostaglandin synthesis, preferably being a non-steroidalanti-inflammatory drug (NSAID), for example, diclofenac, indomethacin,naproxen, and (+/-) tromethamine salt of ketorolac (sold under thetrademark Toradol™) and an effective non-toxic dosage amount (forexample in excess of 5 mg per cm² (square centimeter) of skin or exposedtissue to which the dosage amount of the composition is to be applied)of hyaluronic acid and/or salts thereof (for example the sodium salt)and/or homologues, analogues, derivatives, complexes, esters, fragments,and/or sub units of hyaluronic acid (preferably hyaluronic acid and/orsalts thereof) to transport (to faciliate or cause the transport of) thedrug to the site of the pathology and/or trauma of the disease orcondition. These compositions may be applied topically to treat diseasesand conditions of the skin and/or exposed tissue at the site of thetrauma and/or pathology, (for example, basal cell carcinoma, theprecancerous, often recurrent, actinic keratoses lesions, fungallesions, "liver" spots and like lesions (found for the most part in theepidermis), squamous cell tumours, metastatic cancer of the breast tothe skin, primary and metastatic melanoma in the skin, malignanciesand/or tumours in the skin, genital warts (condyloma acuminata),cervical cancer, and HPV (Human Papilloma Virus) including HPV of thecervix, psoriasis (both plaque-type psoriasis and nail bed psoriasis),corns on the feet and hair loss on the head of pregnant women). Theresults of the treatment with suitable dosage amounts taken from thesecompositions (combinations and formulations) have been in some instancesquite dramatic--difficult situations have been successfully treated andresolved.

Furthermore, application of the dosage amounts of the compositions,combinations and formulations are, systemic independent (there is a lackof a blood level of the drug for example NSAID), are quick to penetrateinto the skin to the site of the trauma and/or pathology because theeffective dosage amount of the form of hyaluronic acid transports(facilitates or causes the transport of) the drug (for example NSAID)particularly to the epidermis where the composition, combination orformulation accumulates and remains for prolonged periods. Thecompositions subsequently clear through the lymphatics and are availablefor the treatment of disease and conditions of the lymphatics.

In this regard effective amounts of the form of hyaluronic acid exceedsin the order of about 5 mg per square cm. (cm²) of the area of forexample the skin and/or exposed tissue to which the dosage amounts ofthe composition is to be applied.

Thus, according to another aspect of the invention, Applicants haveprovided topically applicable percutaneous (intracutaneous) penetrating(best targeting the epidermis) systemic independent acting (not actingessentially through the blood) pharmaceutical compositions (combinationsand formulations) comprising a plurality of dosage amounts eachcomprising, together with pharmaceutical excipients suitable for topicalapplication, a therapeutically effective (to treat and to assist toresolve diseases and conditions of the skin and exposed tissue (forexample basal cell carcinoma, the precancerous, often recurrent, actinickeratoses lesions, fungal lesions, "liver" spots and like lesions (foundfor the most part in the epidermis), squamous cell tumours, metastaticcancer of the breast to the skin, malignancies and/or tumours in theskin primary and metastatic melanoma in the skin, genital warts(condyloma acuminata), cervical cancer, and HPV (Human Papilloma Virus)including HPV of the cervix, psoriasis (both plaque-type psoriasis andnail bed psoriasis), corns on the feet and hair loss on the head ofpregnant women), non-toxic (to the patient) dosage amount of a drug forexample which inhibits prostaglandin synthesis, preferably anon-steroidal anti-inflammatory drug (NSAID), for example, diclofenac,indomethacin, naproxen, and (+/-) tromethamine salt of ketorolac (soldunder the trademark Toradol™) and an effective non-toxic amount ofhyaluronic acid and/or salts thereof (for example, the sodium salt)and/or homologues, analogues, derivatives, complexes, esters, fragments,and/or sub-units of hyaluronic acid (preferably hyaluronic acid andsalts thereof) to transport (facilitate or cause the transport of) thedrug (for example NSAID's) rapidly to the site in the skin (for exampleepidermis) and/or exposed tissue of the disease or condition into thetissue to remain there for a prolonged period of time to assist to treatand assist to resolve the disease or condition for example by blockingprostaglandin synthesis.

Effective dosage amounts of the form of hyaluronic acid to facilitate orcause the transport of the drug into the skin and/or exposed tissue bythe form of hyaluronic acid exceeds about 5 mg.-10 mg. in the dosageamount administered (applied and rubbed in) for each 1 cm² of skinand/or exposed tissue area of the disease or condition (for examplebasal cell carcinoma) to which the dosage amount is applied. The dosageamount applicable will depend upon the surface area of the skin and/orexposed tissue in which the condition or disease exists. Thus if thedisease or condition occupies about 0.5 cm², in excess of about 21/2 mgof the form of hyaluronic acid would be used (applied and rubbed in). Inthe same way if the area is 2 cm², the amount of the form of hyaluronicacid preferably exceeds about 10-20 mg of the dosage amount of theformulation or composition applied. Preferred forms of the hyaluronicacid (for example hyaluronic acid and the sodium salt thereof) havemolecular weights less than about 750,000 daltons (for example about150,000 to about 225,000 daltons) to transport the medicine in the skinand/or exposed tissue. While higher molecular weights of the hyaluronicacid and forms thereof may be used to penetrate the skin and/or exposedtissue and transport the medicines or drugs, where the molecular weightof the hyaluronic acid chosen for use is very large, it is preferredthat the form of hyaluronic acid is autoclaved, to break down thehyaluronic acid to fragments of lesser molecular weight or if feasiblediluted to permit administration and ensure no coagulation on or in theskin. Where the molecular weight of the form of hyaluronic acid beingemployed is large, the concentration of the form of the hyaluronic acidin the composition may for example be reduced (for example to less thanabout 3%) dependent on the molecular weight.

The blockage of prostaglandin synthesis by the transported drug (forexample NSAIDS) then unblocks the macrophages and permits themacrophages of the patient proximate the lesion (for example, the basalcell carcinoma) to destroy the lesion or condition. Treatment by dosageamounts of the composition (formulation and/or combination) eliminatesthe condition without recurrence, even where the lesion has recurred anumber of times after unsuccessful treatments according to the priorart.

Other non-steroidal anti-inflammatory drugs (NSAIDS) may be used such asother propionic acid derivatives, Ibuprofen, acetylsalicylic acid,piroxicam and flunixin.

When dosage amounts of such compositions, combinations and formulationsare applied to the site of the disease or condition for example thebasal cell carcinoma of the patient suffering from the basal cellcarcinoma, over a period of time (for example, for a period of 2-4 weeks3 times daily) the basal cell carcinoma is completely resolved anddisappears.

Thus according to another aspect of the invention there is provided apharmaceutical composition from which dosage amounts may be taken forapplication to the skin and/or exposed tissue, the pharmaceuticalcomposition comprising in a form for application to a human a pluralityof dosage amounts of medicine and/or therapeutic agent to treat adisease or condition in a human and a plurality of dosage amounts ofhyaluronic acid and/or salts and/or homologues, analogues, derivatives,complexes, esters, fragments, and/or sub-units of hyaluronic acid suchthat when dosage amounts of the pharmaceutical composition are takenfrom the composition, the amount of the medicine and/or therapeuticagent comprises an effective non-toxic dosage amount of the medicine totreat the disease or condition in the skin and/or exposed tissue in ahuman and the amount of the form of hyaluronic acid in the dosage amountis present in an effective amount to transport (facilitate or cause thetransport of) the medicine and/or therapeutic agent intradermally(percutaneously, intercutaneously, intracutaneously) into the skin(preferably to the epidermis and dermis) and/or exposed tissue of ahuman to the site of a pathology and/or trauma. The effective amount ofthe form of hyaluronic acid has a molecular weight and concentration totransport the medicine (drug) and/or therapeutic agent to the site oftrauma and/or pathology in the skin and/or exposed tissue. In thisregard the preferred amount of the form of hyaluronic acid in eachdosage amount exceeds 5 mg./cm² and preferably the molecular weight isless than about 750,000 daltons, (in one embodiment about 150,000 toabout 225,000 daltons) in some embodiments with a concentration ofbetween about 1 and 3%, preferably concentrations of between about 2 toabout 3% by weight. Where forms of hyaluronic acid are used havinggreater molecular weights, they are preferably cleaved and/or diluted tosmaller concentrations, to facilitate or cause the transport of themedicine and/or therapeutic agent.

According to another aspect of the invention there is provided apharmaceutical composition (for example a gel or cream) from whichdosage amounts may be taken and applied to the skin to treat a diseaseor condition in humans, for example as discussed above, thepharmaceutical composition comprising:

(1) a medicinal and/or therapeutic agent suitable for treating a diseaseor condition in the skin and/or exposed tissue in humans, for example adrug which inhibits prostaglandin synthesis (for example an NSAID); and

(2) hyaluronic acid and/or salts thereof and/or homologues, analogues,derivatives, complexes, esters, fragments, and sub-units of hyaluronicacid, in a form suitable for administration to the skin and/or exposedtissue in humans; characterized in that an effective non-toxic dosageamount comprising components (1) and (2) taken and administered fromsaid composition (i) is available in the skin and/or exposed tissue uponadministration to treat said disease or condition in humans bypenetration at the site to be treated to the site of trauma and/orpathology, and (ii) comprises an effective non-toxic dosage amount ofcomponent (2) effective to transport (facilitate or cause the transportof) component (1) immediately upon administration percutaneously intothe skin (preferably the epidermis) to the site to be treated forexample the site of trauma and/or pathology where it remains for aprolonged time, accumulating there and from which it is discharged viathe lymphatic system.

Therefore according to another aspect of the invention a pharmaceuticalcomposition is provided comprising:

(1) a medicinal and/or therapeutic agent which for example inhibitsprostaglandin synthesis in a therapeutically effective amount to treat adisease or condition of the skin and/or exposed tissue;

and (2) hyaluronic acid and/or salts thereof and/or homologues,analogues, derivatives, complexes, esters, fragments, and subunits ofhyaluronic acid,

characterized in that said composition

(a) is in a dosage form (for example a gel or cream) which is suitablefor administration to the skin and/or exposed tissue;

and (b) is in such an amount and in such form that (i) component (1) isin an effective dosage amount to treat said disease or condition bypenetration at the site of the skin and/or exposed tissue to be treatedfor example the basal cell carinoma and other lesions; and (ii)component (2) is immediately available to transport (facilitate or causethe transport of) component (1) to the site of trauma and/or pathologyto be treated, percutaneously into the skin (or exposed tissue) wherethe composition resides and accumulates for a prolonged period, andwhich component (2) is in an effective non-toxic dosage amount totransport (facilitate or cause the transport of) component (1) uponadministration, percutaneously into the skin or exposed tissue to thesite of the trauma and/or pathology. Preferably the form of hyaluronicacid in the composition comprises hyaluronic acid and/or salts thereof.An effective amount of the form of hyaluronic acid exceeds about 5-10 mgper square centimeter (cm²) of skin and/or exposed tissue to which it isto be applied.

According to another aspect of the invention there s provided the useof:

(1) a medicinal and/or therapeutic agent for example which inhibitsprostaglandin synthesis,

and (2) hyaluronic acid and/or salts thereof and/or homologues,analogues, derivatives, complexes, esters, fragments, and subunits ofhyaluronic acid,

in the manufacture of a pharmaceutical composition or treating a diseaseor a condition (for example those discussed above) of the skin and/orexposed tissue in a therapy wherein dosage amounts taken from thecomposition each comprise:

(1) a therapeutically effective amount of said medicinal and/ortherapeutic agent and

(2) a therapeutically effective amount of the hyaluronic acid and/orsalts thereof and/or homologues, analogues, derivatives, complexes,esters, fragments, and sub-units of hyaluronic acid, the pharmaceuticalcomposition being characterized in that for each dosage amount takenfrom the pharmaceutical composition, the amount of component (2) isimmediately available to transport component (1) percutaneously to thesite of trauma and/or pathology for example into the epidermis where thecomposition accumulates and remains for a prolonged period, at the siteof the skin or exposed tissue to be treated, and component (2) is in aneffective non-toxic amount to transport (facilitate or cause thetransport of) component (1) into the skin or exposed tissue (for exampleinto the epidermis). Preferably component (2) is hyaluronic acid and/orsalts thereof and preferably the dosage amount of component (2) in theamount of the composition taken from the composition (to be taken fromthe composition) and applied to the skin or exposed tissue is a doseamount greater than about 5-10 mg per cm² of skin and/or exposed tissueto which the dosage amount is to be applied.

The pharmaceutical composition will normally include pharmaceuticallycompatible excipients to provide a form for ease of administration tothe skin and/or exposed tissue for transport into the epidermis. Forexample a suitable dosage amount of a gel may be squeezed from a tube asa ribbon of gel "X" cm long (which dosage amount (in the form of theribbon "X" cm long) contains the effective non-toxic dosage amounts ofthe drug and form of hyaluronic acid. Or a dosage amount of creampackaged in a jar may be scooped from the jar by a measuring device orby "two fingers" in a suitable amount (for example in a spoon containinga premeasured volume or an amount about half the "length of thefingers"). Each of the dosage amounts selected comprises the effectiveamounts of drug (for example NSAID) and effective amount of the form ofhyaluronic acid (for example hyaluronic acid and/or salts thereof). Inthis way the patient may "squeeze" or "scoop" or "what have you" theappropriate dosage amount and apply (rub in) the dosage amount onto theskin and/or exposed tissue for transport into the epidermis.

Thus, according to another aspect of the invention, a method of treatinga disease and/or condition of the skin or exposed tissue, for examplebasal cell carcinoma, the precancerous, often recurrent, actinickeratoses lesions, fungal lesions, "liver" spots and like lesions (foundfor the most part in the epidermis), squamous cell tumours, metastaticcancer of the breast to the skin, primary and metastatic melanoma in theskin, malignancies and/or tumours in the skin, genital warts (condylomaacuminata), cervical cancer, HPV (Human Papilloma Virus) including HPVof the cervix, psoriasis (both plaque-type psoriasis and nail bedpsoriasis), corns on the feet and hair loss on the head of pregnantwomen, in a human is provided comprising administering topically tohuman skin and/or exposed tissue an effective non-toxic dosage amount ofa composition comprising, together with pharmaceutical excipientssuitable for topical application to the skin and/or exposed tissue, forexample in the form of a gel or cream (to give the compositiondefinition and form so that specific dosage amounts are easily selectedor taken for administration (for example squeezed from a tube or scoopedfrom a jar and rubbed into the skin or exposed tissue), atherapeutically effective (to treat and to assist to resolve the diseaseor condition for example basal cell carcinoma or other lesion),non-toxic (to the patient) dosage amount of a drug for example whichinhibits prostaglandin synthesis, for example a non-steroidalanti-inflammatory drug (NSAID), for example, diclofenac, indomethacin,naproxen, and (+/-) tromethamine salt of ketorolac (sold under thetrademark Toradol™) and an effective non-toxic dosage amount ofhyaluronic acid and/or salts thereof (for example, the sodium salt)and/or homologues, analogues, derivatives, complexes, esters, fragments,and/or sub-units of hyaluronic acid (preferably hyaluronic acid andsalts thereof) to transport (facilitate or cause the transport of) thedrug (for example NSAID) into the skin or exposed tissue to the site ofthe disease or condition to be treated percutaneously, (to the site oftrauma and/or pathology), for example into the epidermis, where the formof hyaluronic acid and medicine accumulates and remains for a prolongedperiod of time thereby for example blocking prostaglandin synthesis inthe skin or exposed tissue. The form of hyaluronic acid is then clearedthrough the lymphatics (lymphatics system).

Thus, according to another aspect of the invention, the treatment mayemploy the use of the composition, formulation or combination for thetreatment of the diseases and conditions aforesaid as for example byapplying dosage amounts of the composition, formulation or combination anumber of times daily (for example, 3 times daily) for a period of time,for example, 2-4 weeks to clear the disease, lesion or condition. Eachdosage amount applied will depend upon the size of the lesion orcondition on the skin or exposed tissue. For example, a suitable dosageamount may include 5-10 mg. of the form of hyaluronic acid per 1 cm²skin area or exposed tissue area.

One such formulation may comprise 3% (by weight) diclofenac in a 21/2%(by weight) hyaluronic acid (sodium hyaluronate--molecular weight661,600) gel formulation, with the excipients being glycerine (5%),benzyl alcohol (3%) (acting in part as a solubilizer and preservative),and sterile water (the balance) in a 50 gm. tube of the composition (aplurality of dosage amounts) whose tube O.D. (outer diameter) of theopening through which the gel formulation is discharged from the tube is8 mm and whose I.D. (inner diameter) of the opening is 4 mm. Therefore aribbon 2-3 cm in length, squeezed from a tube gives about 5 mg-71/2 mgof hyaluronic acid for application to a skin or exposed tissue surfacearea of 1-11/2 cm² with an effective dosage amount of diclofenac. Whilegreater amounts squeezed from the tube, may be applied, the applicationof substantial excessive dosage amounts to the skin and/or exposedtissue may saturate the skin or exposed tissue and thus the epidermis.(There is therefore no more room for the composition to pass between thecells and therefore further applications at that time will not provideadditional benefit). Where pain relief is also required additionaldosage amounts, for example in excess of about 10 mg. of the hyaluronicacid taken from the same pharmaceutical composition applied per/cm² ofsurface area of the skin or exposed tissue may be required to beapplied.

Another formulation may comprise 3% (by weight) diclofenac in a 21/2%(by weight) hyaluronic acid (sodium hyaluronate--molecular weight679,000) gel formulation (also in a tube) with excipients being benzylalcohol (1%) (a preservative), methoxypolyethylene glycol 350 (20% byweight) (a solubilizer), and sterile water (the balance).

While the above compositions, combinations and formulations areproposed, provided there is sufficient amounts of the form of thehyaluronic acid (for example, sodium hyaluronate) in the dosage amountsapplied to the skin and/or exposed tissue to facilitate or cause thepercutaneous (intracutaneous) transport of the drug for example whichinhibits prostaglandin synthesis, preferably an NSAID (for example,diclofenac) to block prostaglandin synthesis, then the formulations maybe of any suitable form, for example, a 1% lotion of hyaluronic acidwith NSAID, or a cream or gel or any other suitable form.

Therefore according to another aspect of the invention, there isprovided containers (for example tubes and jars) containing compositionscomprising a plurality of dosage amounts of the drug and form ofhyaluronic acid, each dosage amount comprising an effective non-toxicdosage amount of the drug and an effective non-toxic dosage amount ofthe form of hyaluronic acid (preferably sodium hyaluronate havingmolecular weight less than about 750,000 daltons) to transport the druginto the skin and/or exposed tissue. In some embodiments, means areprovided to assist the removal from the container of an effective dosageamount of the composition in the container for use to apply to the skinor exposed tissue at the site of trauma and/or pathology to treat thedisease and/or condition (for example mouth opening of a tube to controlthe amount discharged from the tube).

Furthermore, because there is little concern with respect to thetoxicity or adverse effects of the use of, for example, the NSAIDs withthe hyaluronic acid in the compositions of this invention the NSAID maybe combined as needed (after solubilizing (if required) of the NSAID ina suitable solubilizer) with the form of the hyaluronic acid.

Therefore according to another aspect of the invention, percutaneous(intercutanous) delivery of a therapeutically effective dosage amount ofa drug (in a composition, combination or formulation) and which drug forexample inhibits prostaglandin synthesis, preferably being anon-steroidal drug (NSAID) is provided. In this regard the drug istransported to the site of, on, or in the skin and/or exposed tissue oftrauma and/or pathology to treat the disease or condition for examplethe basal cell carcinoma or actinic keratoses lesion in a mammal(human). The delivery may comprise topically administering (to the skinor exposed tissue site of for example the basal cell carcinoma or otherlesion) a therapeutically effective non-toxic (to the patient) dosageamount of a composition comprising a drug for example which inhibitsprostaglandin synthesis, preferably an NSAID (non-steroidalanti-inflammatory drug), for example, diclofenac, indomethacin,naproxen, and (+/-) tromethamine salt of ketorolac (sold under thetrademark Toradol™), and an effective non-toxic amount of hyaluronicacid and/or salts thereof and/or homologues, analogues, derivatives,complexes, esters, fragments, and sub-units of hyaluronic acid,preferably hyaluronic acid and salts thereof, sufficient to transport,(facilitate or cause the transport of), the drug for example NSAIDpercutaneously (to for example the epidermis) to the site of the traumaand/or pathology in for example the epidermis, for example the basalcell carcinoma (or other lesion), to be treated for example to block thesynthesis of prostaglandins.

Delivery may be also accomplished by the same amount of the form ofhyaluronic acid, of other drugs percutaneously (intercutaneously) to theskin and exposed tissue by application and rubbing in of an effectivenon-toxic dosage amount of the formulation or composition comprising aneffective non-toxic dosage amount of the drug and an effective non-toxicdosage amount of the form of hyaluronic acid for the transport of thedrug percutaneously into the skin or exposed tissue to the epidermiswhere the dosage amount of the composition is accumulated and remainsfor a prolonged period of time before the form of hyaluronic acid iscleared through the lymphatics. In this regard the drug may benovantrone (an anti-cancer drug) for administration to a tumour ormalignancy in the skin. The novantrone may comprise 10 mg in the dosageamount of the composition and the form of hyaluronic acid may be inexcess of about 5 mg of sodium hyaluronic per cm² of the skin or exposedtissue (about 2.5% of the composition) for the percutaneous transport ofthe novantrone.

Thus, according to another aspect of the invention, use of acomposition, combination or formulation is provided to treat a diseaseor condition for example basal cell carcinoma (or other lesion), by theapplication of the composition, combination or formulation, the amountof the composition, combination and formulation administered comprisingtogether with pharmaceutical excipients suitable for topicalapplication, a therapeutically effective (to treat and assist to resolvea disease or condition for example, basal cell carcinoma), non-toxic (tothe patient) amount of a drug for example which inhibits prostaglandinsynthesis preferably a non-steroidal anti-inflammatory drug (NSAID), forexample, diclofenac, indomethacin, naproxen, and (+/-) tromethamine saltof ketorolac (sold under the trademark Toradol™) administered togetherwith, or carried in, an effective dosage amount of hyaluronic acidand/or salts thereof (for example, the sodium salt) and/or homologues,analogues, derivatives, complexes, esters, fragments, and/or sub-unitsof hyaluronic acid (preferably hyaluronic acid and salts thereof)effective to transport the drug for example the NSAID (to facilitate orcause the transport of the drug for example NSAID) percutaneously intothe skin especially the epidermis at the site of the disease orcondition for example basal cell carcinoma (or other lesion) to betreated, thereby, if an NSAID, blocking prostaglandin synthesis toenable the macrophages (and N.K. cells) to resolve the disease orcondition for example basal cell carcinoma or other lesion.

Applicants postulate that the hyaluronic acid and/or salts thereofand/or the homologues, analogues, derivatives, complexes, esters,fragments, and/or sub units of hyaluronic acid facilitate or cause thetransport of the drug for example which blocks prostaglandin synthesis(preferably an NSAID) to the site of prostaglandin synthesis to blockprostaglandin synthesis.

Applicants' compositions and dosage amounts of their compositions andthe use of their compositions and dosage amounts of their compositions,at the same time, abate pain that the patient is experiencing at thepaccinian nerve bundles (superficial nerve bundles) at the site of thetrauma and/or pathology on/in the exposed tissue and/or skin.

Thus, according to another aspect of the invention, a method of abatingpain in the skin and/or exposed tissue for example suffering a diseaseor condition (for example those discussed above), and a composition fromwhich dosage amounts may be taken and applied (rubbed in) which isuseful for abating such pain are provided, the method comprisingadministering (rubbing on) an effective dosage amount of the compositionto the skin and/or exposed tissue, and the composition comprises aplurality of dosage amounts, each comprising an effective non-toxicdosage amount of an NSAID and an effective non-toxic dosage amount ofthe hyaluronic acid and/or salts thereof and/or homologues, analogues,derivatives, complexes, esters, fragments, and/or subunits of hyaluronicacid (preferably hyaluronic acid and salts thereof), for example amountsexceeding 10-20 mg. per square cm (cm²) of skin or exposed tissue towhich it is applied, for percutaneous transport of the NSAID by the formof hyaluronic acid into the epidermis proximate the paccinian nervebundles (superficial nerve bundles at the end of the nerves) to abatepain. Thus, according to another aspect of the invention, compositionsare provided for use to relieve pain from which dosage amounts of thecomposition comprising dosage amounts of the NSAID and form ofhyaluronic acid are taken.

By way of example and to illustrate the facilitation of the delivery ortransport of a chemical to a site in a human, when ethyl alcohol isinjected directly into a tumour and sonographic (ultrasound) assessmentis made, it is not dispersed throughout the tumour. When the ethylalcohol to be administered into a tumour is carried by hyaluronic acidand/or salts thereof, sonographic assessment of the tumour demonstratesthe dispersion of the ethyl alcohol throughout the tumour.

While Applicants postulate that the hyaluronic acid facilitate or causesthe transport and delivery, Applicants' invention may be used asdescribed irrespective of the actual method of operation of thehyaluronic acid and/or salts thereof and/or the homologues, analogues,derivatives, complexes, esters, fragments and sub-units of hyaluronicacid.

The combination of hyaluronic acid and salts thereof and other formswith drugs for example that inhibit prostaglandin synthesis, for exampleNSAIDs, alters their distribution and performance in the skin and/orexposed tissue particularly the epidermis (the combinations andformulations being systemic independent), and produces an unusualtargeting for underperfused skin and/or pathological tissue in the skin(site of trauma and/or pathology). The application may be made asrequired with the amount depending upon the condition of the skin orexposed tissue.

As a major amount of soluble indomethacin may be incorporated into theformulation, or composition, the indomethacin may be solubilized usingn-methyl glucamine at a dilution of 5 mg/ml of n-methyl glucamine (NMG).This substance is then passed through a 22 micron Milipore filter toproduce sterility. This material is non-toxic at 16 fold the therapeuticdose in animals (with hyaluronic acid) and for this reason wasconsidered appropriate to be used in human conditions. Thus, Indocid™solubilized in NMG may be administered with hyaluronic acid topicallyfor percutaneous penetration at, for example, varying doses. Thesolution of indomethacin and NMG may be mixed with, for example,"LifeCore™" hyaluronic acid in dosage amounts discussed above. Thisproduces an appropriate mixture and can be administered safely.

When the NSAID, for example indomethacin (dissolved in n-methylglucamine) or other NSAID, is applied topically in an effective dosageamount from a composition or formulation also including the effectivedosage amount of the form of hyaluronic acid, no major toxic sideeffects occur, such as gastro-intestinal distress, neurologicalabnormalities, depression, etc., even at elevated amounts ofindomethacin (if necessary). (This may be in part because of theclearing of the hyaluronic acid through the lymphatic system from thesite). In addition, the responses that have been observed are dramaticwhen the drug for example NSAID (for example diclofenac) is combinedwith hyaluronic acid, demonstrating clearly that the combination is now"targeting" to the site of pathology or trauma, or pathological tissue.Furthermore, patients using the formulations and combinations of drug(for example NSAID)--hyaluronic acid (sodium hyaluronate) (for example,diclofenac or indomethacin and hyaluronic acid), experience dramaticrelief of pain immediately.

Thus, Applicants believe that the use of the NSAID, for example withhyaluronic acid (sodium hyaluronate), deblocks the macrophages (and N.K.cells (Natural Killer Cells) thought to be immature macrophages) bypreventing enzymatic production of prostaglandin which blocks macrophage(and N.K. cell) functioning. The hyaluronic acid (and salt and otherforms) not only enhances the activity of the drug (NSAID) but alsoreduces any side effects and toxicity that is associated with the use ofthe prostaglandin synthesis inhibitors. When effective dosage amounts ofcompositions, formulations and combinations containing effective dosageamounts of the drugs for example, (NSAIDs (for example, diclofenac)) andeffective dosage amounts of, for example, hyaluronic acid or the sodiumsalt thereof, are applied to for example the tumour lesion (for examplebasal cell carcinoma) or other condition (for example, actinic keratoseslesion) for a period of time (for example, 3 times daily for 2-4 weeks),the carcinoma and lesions, as the case may be, disappear.

Applicants also postulate that when the combination or formulation isapplied to the disease or condition (for example, basal cell carcinomaor actinic keratoses), the hyaluronic acid passes between the cells (inthe stratum corneum and epidermis to the dermis depending on amounts) tothe areas of trauma and/or pathology deficient in hyaluronic acid (orforms thereof), transporting, taking, drawing, carrying or pulling theNSAID with it to the sites of prostaglandin synthesis, penetrating toinhibit prostaglandin synthesis until the space between the cells issaturated. The NSAID now being proximate the Paccinian nerve bundle(superficial nerve bundles at the end of the nerves) gives pain relief.The macrophages (which had been previously blocked) are unblocked andact to destroy the disease or condition for example basal cellcarcinoma, actinic keratoses lesion, or other disease or lesion.Furthermore, the effective non-toxic dosage amount of the composition,combination or formulation, comprising the effective dosage amount ofthe form of hyaluronic acid and the effective dosage amount of NSAIDpassing through the stratum corneum to the epidermis and to the dermis(if a sufficient amount of the form of hyaluronic acid is present),passes into the skin, accumulating and staying longer in the skin at thesite of the trauma and/or pathology. Therefore, after having had animmediate effect at the site of trauma and/or pathology (for example,relieving pain and acting on the basal cell carcinoma, actinic keratosesand other disease, condition or lesion), the NSAID-hyaluronic acidcombination continues to accumulate at the site in need of treatment andthereafter clears through the lymphatic system.

Thus according to another aspect of Applicant's invention, Applicants'compositions, formulations and combinations quickly penetrate onapplication through the stratum corneum into the epidermis (to thedermis) by the form of hyaluronic acid transporting the NSAID, to thesite of trauma and/or pathology where the amounts applied accumulate andremain for a prolonged time for treatment.

Fifteen (15) minutes after application of one of Applicants'formulations, about three times the amount of Applicants' formulationhas penetrated into the skin (particularly the epidermis) thanformulations and combinations not containing hyaluronic acid oreffective dosage amounts of hyaluronic acid, but containing the samedrug. Furthermore, the drug and hyaluronic acid accumulate and remain atthe site in need of treatment for a longer period of time.

Thus according to another aspect of the invention, non-toxic effectivedosage amounts of forms of hyaluronic acid (preferably sodiumhyaluronate) and effective non-toxic dosage amounts of a drug may beadministered in compositions to sites of trauma or pathology, on/in theskin and/or exposed tissue (for example the epidermis) by theapplication of the effective non-toxic dosage amount of the compositioncomprising an effective non-toxic dosage amount of a drug (for examplean NSAID) and an effective non-toxic dosage amount of a form ofhyaluronic acid (for example sodium hyaluronate) to the skin or exposedtissue whereby the forms hyaluronic acid transport the drugpercutaneously to the site of trauma and/or pathology where thecomposition accumulates and remains for a prolonged period of timethereby retaining the drug at the site of trauma and/or pathology (forexample the epidermis) for the treatment of the condition or disease andthe reduction of pain.

Thus according to another aspect of the invention, Applicants haveprovided compositions (formulations and combinations) (includingpharmaceutical excipients suitable for topical application) from whicheffective non-toxic (to the patient) dosage amounts of a drug (forexample an NSAID) to treat and to assist to resolve diseases andconditions of the skin and/or exposed tissue (for example basal cellcarcinoma, the precancerous, often recurrent, actinic keratoses lesions,fungal lesions, "liver" spots and like lesions (found for the most partin the epidermis), squamous cell tumours, metastatic cancer of thebreast to the skin, primary and metastatic melanoma in the skin,malignancies and/or tumours of the skin, gential warts, cervical cancer,and HPV (Human Papilloma Virus) including HPV of the cervix, psoriasis(both plaque-type psoriasis and nail bed psoriasis), corns on the feetand hair loss on the head of pregnant women), and effective non-toxicdosage amounts of hyaluronic acid and/or salts thereof (for example, thesodium salt) and/or homologues, analogues, derivatives, complexes,esters, fragments, and/or sub-units of hyaluronic acid (preferablyhyaluronic acid and salts thereof) sufficient to transport (tofacilitate or cause the transport of) the drug, for example NSAID, aretaken for application, to a site in the skin (for example epidermis) orexposed tissue having a disease or condition for percutaneous transportinto the skin and/or exposed tissue to accumulate and remain there for aprolonged period of time to for example block prostaglandin synthesis.Thus an effective dosage amount of the composition or formulation orcombination penetrates quickly into the skin, for example by thehyaluronic acid transporting the NSAID or causing the NSAID to betransported for example to the epidermis of the skin, accumulates thereand remains there for a prolonged period of time, thereby accumulatingthe drug and forms of hyaluronic acid in the skin (particularly theepidermis).

Thus according to another aspect of the invention, a method ofaccumulating a drug and a form of hyaluronic acid in skin and/or exposedtissue is provided comprising topically administering a therapeuticallyeffective non-toxic dosage amount of a composition comprisingpharmaceutical excipients suitable for topical applications, aneffective non-toxic (to the patient) dosage amount of a drug for examplewhich inhibits prostaglandin synthesis, preferably a non-steroidalanti-inflammatory drug (NSAID), for example, diclofenac, indomethacin,naproxen, and (+/-) tromethamine salt of ketorolac (sold under thetrademark Toradol™) (to treat and to assist to resolve the disease andconditions of the skin and exposed tissue (for example basal cellcarcinoma, the precancerous, often recurrent, actinic keratoses lesions,fungal lesions, "liver" spots and like lesions (found for the most partin the epidermis), squamous cell tumours, metastatic cancer of thebreast to the skin, malignancies and/or tumours of the skin, primary andmetastatic melanoma in the skin, genital warts cervical cancer, and HPV(Human Papilloma Virus) including HPV of the cervix, psoriasis (bothplaque-type psoriasis and nail bed psoriasis), corns on the feet andhair loss on the head of pregnant women), and an effective non-toxicdosage amount of hyaluronic acid and/or salts thereof (for example, thesodium salt) and/or homologues, analogues, derivatives, complexes,esters, fragments, and/or sub-units of hyaluronic acid (preferablyhyaluronic acid and salts thereof) effective to transport (to facilitateor cause the transport of) the drug (for example NSAID) percutaneouslyto the site in the skin (for example epidermis) or exposed tissue of thedisease or condition to accumulate and remain there for a prolongedperiod of time for example to block prostaglandin synthesis.

According to another aspect of the invention, a method of quicklydelivering a drug to the skin or exposed tissue, particularly theepidermis, and maintaining the drug therein for a prolonged period oftime is provided, the method comprising topically administering (forexample rubbing in) an effective non-toxic dosage amount of acomposition comprising pharmaceutical excipients suitable for topicalapplication, a therapeutically effective (to treat and assist to resolvethe disease and/or condition of the skin and exposed tissue (for examplebasal cell carcinoma, the precancerous, often recurrent, actinickeratoses lesions, fungal lesions, "liver" spots and like lesions (foundfor the most part in the epidermis), squamous cell tumours, metastaticcancer of the breast to the skin, primary and metastatic melanoma in theskin, malignancies and/or tumours of the skin, genital warts, cervicalcancer, and HPV (Human Papilloma Virus) including HPV of the cervix,psoriasis (both plaque-type psoriasis and nail bed psoriasis), corns onthe feet and hair loss on the head of pregnant women)), non-toxic (tothe patient) dosage amount of a drug for example which inhibitsprostaglandin synthesis, preferably a non-steroidal anti-inflammatorydrug (NSAID), for example, diclofenac, indomethacin, naproxen, and (+/-)tromethamine salt of ketorolac (sold under the trademark Toradol™) andan effective non-toxic dosage amount of hyaluronic acid and/or saltsthereof (for example, the sodium salt) and/or homologues, analogues,derivatives, complexes, esters, fragments, and/or sub-units ofhyaluronic acid (preferably hyaluronic acid and salts thereof)sufficient to transport (to facilitate or cause the transport of) thedrug for example the NSAID percutaneously to the site of the traumaand/or pathology in the skin (for example epidermis) or exposed tissue,for remaining there for a prolonged period of time (for example in theepidermis and dermis) to for example block prostaglandin synthesis.Suitable amounts of the form of hyaluronic acid may comprise in excessof 5 mg. per cm² in a form which transports the drug (for examplemolecular weights of the form of hyaluronic acid being less than about750,000 Daltons or if at substantially greater molecular weights,diluted (to reduce) the concentration or autoclaved or cleaved ifrequired to reduce the size of the molecules.

According to another aspect of the invention, a method of controllingthe unloading of a drug from the skin or exposed tissue into thelymphatic system comprises delivering (transporting) an amount of druginto the skin or exposed tissue by an effective non-toxic dosage amountof a form of hyaluronic acid and/or salts thereof and/or homologues,analogues, derivatives, complexes, esters, fragments, and/or sub-unitsof hyaluronic acid to the skin (epidermis) or exposed tissue to controlthe unloading of the drug into the lymphatic system (for example by theapplication of greater than 5 mg./cm²) of the form of hyaluronic acid.

Thus according to another aspect of the invention a composition isprovided which when administered to a human by preferably administrationto the skin and/or exposed tissue of a human, unloads its contents intothe lymphatic system, the composition comprising an effective non-toxicdosage amount of a drug (for example an NSAID or an anti-cancer drug(Novantrone) and an effective non-toxic amount of hyaluronic acid and/orsalts thereof and/or homologues, analogues, derivatives, complexes,esters, fragments and/or sub-units of hyaluronic acid (for example atleast about 5-10 mg/cm² of skin or exposed tissue). Thus the compositionis made up of a plurality of such dosage forms (for example a cream orlotion or gel).

Thus according to another aspect of the invention, a new composition fortreating diseases via the lymphatic system is provided comprising aplurality of effective non-toxic dosage amounts of the composition, eachdosage amount comprising hyaluronic acid and/or salts thereof and/orhomologues, analogues, derivatives, complexes, esters, fragments and/orsub-units of hyaluronic acid for passing into the lymphatic system and atherapeutic effective amount of medicine for treatment of a disease(which disease may be in the lymphatic system).

According to another aspect of the invention, the composition may be forapplication to the skin or exposed tissue.

According to another aspect of the invention, a composition is providedfrom which effective dosage amounts may be taken and administered, eacheffective dosage amount of the composition comprising an effectivenon-toxic dosage amount of hyaluronic acid and/or salts thereof and/orhomologues, analogues, derivatives, complexes, esters, fragments and/orsub-units for transporting a therapeutically effective non-toxic dosageamount of a medicine and/or therapeutic agent (for example an NSAID) inthe composition into the skin and/or exposed tissue when applied theretoto an area of pathology and/or trauma then into the lymphatic system,the dosage amount being essentially systemic independent such thatsubstantial amounts do not enter the blood system prior to clearing(passing) into the lymphatic system. Preferably the amount of the formof hyaluronic acid in each dosage amount administered is greater thanabout 5-10 mg./cm² and the molecular weight is less than about 750,000daltons.

We have compared the penetration and retention of one of ourcombinations (formulations), with a control and Voltarol Emulgel in theskin as follows:

    ______________________________________                                        (A) OUR FORMULATION                                                           1% DICLOFENAC IN 3.0% HA GEL 50 g/tube                                        EPDICLO1                                                                      LOT XPB 044  Quantity 1500 ml                                                 FORMULA    Supplier Lot      Amount    Percent                                ______________________________________                                        Sterile Water                                                                            Baxter   AW45F1   1397 ml     --                                   Glycerin   Life     1043     45   g (36 ml)                                                                            3%                                   Benzyl Alcohol                                                                           Caledon  02517    22.5 g (22 ml)                                                                              1.5%                               Liquid Wax DICDD                                                                         Brooks   191-175  45   g      3%                                   Diclofenac Sodium                                                                        Prosintex                                                                              9113003  15   g      1%                                   Sodium Hyaluronate                                                                       Skymart  HG-1103  45   g      3%                                   ______________________________________                                         Mol. Wt. 661,600                                                         

PROCEDURE

Set up stirring apparatus using a 3 liter stainless steel beaker

Add Water, Glycerin, Benzyl Alcohol and Liquid Wax DICDD, stir and mixfor 10 minutes

Add Diclofenac Sodium and stir for 30 minutes to dissolve

Add Sodium Hyaluronate and stir for 90 minutes

FILLED

In a 50 ml aluminum collapsible tube, inside of tube lacquered with aphanolic resin, outside of tube white regular enamel coating;

9 mm white polypropylene screw on cap with pierce tip

    ______________________________________                                        Gels               Batch No.s                                                 ______________________________________                                        (B) Voltarol Emulgel   060400 10 93                                           (C) 1% Diclofenac Gel  XPB049 (Control)                                       ______________________________________                                        (C) CONTROL                                                                   1% DICLOFENAC IN CARAPOL GEL, 50 g Jar                                        LOT XPB 049 Quantity 100 ml                                                   FORMULA    Supplier    Lot      Amount                                                                              Percent                                 ______________________________________                                        Sterile Water                                                                            Baxter      AW45N5   93  ml  --                                    Glycerin   BDH         2579     3   g   3%                                    Benzyl Alcohol                                                                           BDH         23797    1.5 g     1.5%                                Liquid Wax DICDD                                                                         Brooks      L-1424   3   g   3%                                    Diclofenac Sodium                                                                        Prosintex   9113003  1   g   1%                                    Carbopol 934                                                                             A&C Chemicals                                                                             910304   1   g   1%                                    ______________________________________                                    

PROCEDURE

Set up stirring apparatus using a 400 ml stainless steel beaker

Add Water, Glycerin, Benzyl Alcohol, Liquid Wax DICDD, and stir to mixthoroughly for 10 minutes

Add Diclofenac Sodium and stir for 20 minutes to dissolve

Very slowly add Carbopol 934, avoid getting lumps

    ______________________________________                                        Samples                                                                                            Quantity of gel applied                                  Cell      Sample     (mg)                                                     ______________________________________                                        A         060400 10 93                                                                             192                                                      B         060400 10 93                                                                             192                                                      C         EPDICLO1*  192                                                      D         EPDICLO1*  192                                                      E         XPB049     192                                                      F         XPB049     192                                                      ______________________________________                                         *  Our Formulation                                                       

Skin Type

One piece of skin (Female, 37 years, smoker, breast skin) was used forone sample from each batch. A second piece of skin (no further detailsavailable) was used for the second sample from each batch. The skin wasstored deep frozen (→20° C.) until thawed for this experiment. Fullthickness skin was used for this experiment.

Experimental Conditions

Skin permeation cells were prepared containing an exposed skin surfacearea of 9.6 cm² and a constantly stirred receptor fluid beneath the skinconsisting of 135 ml of ethanol:phosphate buffered saline (25:75 v/v).

Each cell was allowed to equilibrate for 1 hour at 37° C. after whichthe gel was spread evenly over the skin surface at a concentration of 20mg/cm²). See table above. The cell was then maintained at 37° C. with anair temperature above the skin of 35° C.

24 hours after application of the gel the experiment was stopped and aportion of the receptor fluid removed. The skin was removed from thecell and any gel remaining on the surface carefully wiped off with drypaper towel followed by paper towel moistened with water. The skin wascut with a scalpel to obtain thin top and thicker lower sections ofskin.

This was done in order to obtain layers of skin which approximated theepidermal and dermal layers. Each skin section was weighed and theresidual diclofenac extracted with 10 ml of fresh receptor fluid usingan ultra turrax homogeniser. The homogenates were centrifuged and aportion of the resultant supernatant solutions removed.

The receptor fluid and skin extracts from each cell were assayed fordiclofenac content by using a validated reverse phase high performanceliquid chromatography (HPLC) method.

Results

    ______________________________________                                        Distribution of Diclofenac 24 hours after application of                      Diclofenac Gel                                                                           Top Skin portion                                                          Recep-                                                                              Skin              Bottom skin portion                                     tor     Weight            Skin                                       Sample   μg   (g)     μg                                                                              μg/g                                                                            Weight                                                                              μg                                                                              μg/g                         ______________________________________                                        (Voltarol                                                                     Emugel)                                                                       060400 10 93                                                                           447     0.1363  101  742  1.2449                                                                              217  174                             060400 10 93                                                                           764     0.2445  141  577  1.2351                                                                              202  164                             Mean     606                  660             169                             (Our                                                                          Formulation)                                                                  EPDICLO1 247     0.1535  133  867  1.4663                                                                              148  101                             EPDICLO1 292     0.1647  145  879  1.0022                                                                               86  86                              Mean     269                  873             93                              (Control)                                                                     XPB049   184     0.1275   35  272  1.1324                                                                               58  51                              XPB049   147     0.2068   82  396  1.0893                                                                               68  63                              Mean     165                  334             57                              ______________________________________                                    

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 illustrates the presence of diclofenac in the top skin portionafter administration of the several compositions.

FIG. 2 illustrates the presence of diclofenac in the bottom skin portionafter administration of the several compositions.

FIG. 3 illustrates the presence of diclofenac in the receptor solutionafter the elapse of a predetermined time.

FIG. 4 illustrates the results of the anti-cancer agent 5-fluoracil whenadministered alone and when administered with hyaluronic acid at varyingtimes for tumor skin and normal skin, respectively.

FIG. 5 illustrates the radioactivity in tumor tissue.

FIGS. 6a and 6b show the response of basal cell carcinoma in an 82 yearold male patient to treatment with NSAIDS and hyaluronic acid gel.

FIGS. 7a and 7b show the response of basal cell carcinoma in a 45 yearold male patient to treatment with NSAIDS and hyaluronic gel.

FIGS. 8a-8d show the response of basal cell carcinoma in an 82 year oldmale patient to treatment with NSATDS and hyaluronic gel.

FIGS. 9a-9d show the response of basal cell carcinoma in a 64 year oldmale patient to treatment with NSAIDS and hyaluronic gel.

FIGS. 10a-10d show the response of basal cell carcinoma in an 86 yearold male patient to treatment with NSAIDS and hyaluronic gel.

FIGS. 11a-11c show the response of basal cell carcinoma in a 70 year oldfemale patient to treatment with NSAIDS and hyaluronic gel.

FIGS. 12a and 12b show the response of p815 tumors in mice (strain DBA₂)to treatment with novantrone and hyaluronic gel.

DESCRIPTION OF EMBODIMENTS

Thus having regard to the above and FIGS. 1, 2 and 3, it is clear thatthe sodium hyaluronate takes the diclofenac into the skin to theepidermis level (See FIG. 1) more rapidly than the Voltarol Emugel ornon-hyaluronic acid diclofenac containing control formulation,accumulates it there and retains it there longer. The other formulationspermit the NSAID, diclofenac, to pass through the bottom skin portion(dermis) quicker, thereby clearing it from the epidermis and dermis,quicker. Furthermore, more of Applicants' formulation is in theepidermis and in the dermis even after 12 hours. With respect to FIG. 1,the top of the graph should have the following heading "DICLOFENAC TOPSKIN PORTION", the left side of the graph should have the following sideheading "DICLOFENAC (MICROGRAMS) (THOUSANDS)" and the bottom of thegraph should have the following bottom heading "ELAPSED TIME(HOURS)□0604001093+EPDICLO1⋄XP8049". With respect to FIG. 2, the top ofthe graph should have the following heading "DICLOFENAC BOTTOM SKINPORTION", the left side of the graph should have the following sideheading "DICLOFENAC (MICROGRAMS)" and the bottom of the graph shouldhave the following bottom heading "ELAPSED TIME(HOURS)□0604001093+EPDICLO1⋄XP8049". With respect to FIG. 3, the top ofthe graph should have the following heading "DICLOFENAC RECEPTORSOLUTION", the left side of the graph should have the following sideheading "DICLOFENAC (MICROGRAMS)" and the bottom of the graph shouldhave the following bottom heading "ELAPSED TIME(HOURS)□0604001093+EPDICLO1⋄XP8049".

It is also clear that Applicants' formulations clear into the lymphaticsystem not through the blood system. Yet the prior art topicalformulations have always tried "to drive" the formulations through theskin into the blood for treatment of the disease or condition in thearea (i.e. systemic action).

Thus, our composition, formulation and combination, (and dosage amountsthereof) penetrate quickly and rapidly at the site of treatment throughthe upper skin into the epidermis, where the paccinian bundles arelocated and the NSAID and the form of hyaluronic acid are accumulatedand are retained longer, where needed (for example for the treatment ofbasal cell carcinoma).

Further, the NSAIDs are retained in the area to be treated with the formof hyaluronic acid. In doing so, they preclude prostaglandin synthesis,in effect, deactivating the synthesis or inhibiting the synthesis, ofprostaglandins, permitting the macrophages' scavenger cell activity toeliminate the tumour and lesion. Additionally, a rapid onset of painrelief (analgesic effect) is provided (depending on the amount of NSAIDand form of hyaluronic acid) usually where in excess of about 10 mg ofthe form of hyaluronic acid (preferably hyaluronic acid and saltsthereof) is administered per cm² of surface area comprises the dosageamount administered. However, there are no blood levels of the NSAID inthe immediate area of treatment. The forms of hyaluronic acid are thuscleared via the lymphatic system. Then the lymphatics pass the forms ofhyaluronic acid, Applicants believe, to the blood system. Thus, theNSAIDs and forms of hyaluronic acid stay at the site to be treated forwell in excess of 12-24 hours, a protracted stay.

Thus, over the period of treatment (for example, applications ofeffective non-toxic dosage amounts of compositions containing forexample effective non-toxic dosage amounts of the NSAIDS and effectivenon-toxic dosage amounts of the sodium hyaluronate, 3 times a day for2-4 weeks, transport the NSAIDS to to the epidermis to inhibitprostaglandin synthesis to enable the macrophages to "scavenge" thetumour cells and eliminate them. The end result is the successfultreatment of the disease or condition at the site of trauma and/orpathology of the skin or exposed tissue, for example, the resolution of,the basal cell carcinoma, the precancerous, often recurrent, actinickeratoses lesions, fungal lesions, "liver" spots and like lesions (foundfor the most part in the epidermis), squamous cell tumours, metastaticcancer of the breast to the skin, malignancies and/or tumours in theskin, primary and metastatic melanoma in the skin, genital wartscervical cancer, and HPV (Human Papilloma Virus) including HPV of thecervix, psoriasis (both plaque-type psoriasis and nail bed psoriasis),corns on the feet and hair loss on the head of pregnant women, withcomplete disappearance of the disease or condition as the case may be,by topical therapy without resorting to surgery.

One of the formulations which we have employed successfully is a gelformulation comprising 3% diclofenac in 2.5% sodium hyaluronateformulated as follows:

    ______________________________________                                        Formulation 1 (3000 ml.)                                                      Formula    Supplier (LOT)    Amount    Percent                                ______________________________________                                        Glycerine  Life     1043     150 g (119 ml)                                                                          5                                      Benzyl Alcohol                                                                           Caledone 02517    90 g (86 ml)                                                                            3                                      Diclofenac Sodium                                                                        Prosintex                                                                              9113003  90 grams  3                                      Sodium Hyaluronate                                                                       Skymark  HG1003   75 grams  2.5                                    (MW 661,660)                                                                  Sterile water                                                                            Baxter   AW4455   2795 ml.                                         balance                                                                       ______________________________________                                    

Procedure

set up stirring apparatus using a 4 liter stainless steel beaker

add water, Glycerine, and Benzyl Alcohol; stir to mix

add Diclofenac Sodium and stir for 30 minutes

then add the Sodium Hyaluronate and stir for 90 minutes

initially, stir at a high torque but avoid splashing; as the gelthickens, stir at a lower torque.

The gel is then packaged in a tube or jar or other suitable containerfor use. Identification of suitable dosage amounts and how they aretaken from the container may be provided with the container--for examplesqueeze "X" cm. of ribbon from the tube; fill spoon or spatulaaccompanying jar; (the spoon or spatula containing a predetermineddosage amount) then apply and rub into site of trauma and/or pathology(the dosage amount indicated will be such amount of the compositionwhich comprises in excess of about 5 mg. of sodium hyaluronate per cm²(square centimeter) of skin or exposed tissue to which the dosage amountis to be applied. The amount of Diclofenac Sodium was determined in thesame manner (having regard to the dosage amount required).

Another such formulation is:

    ______________________________________                                        Formulation 2                                                                 Formula      Supplier (LOT)    Amount  Percent                                ______________________________________                                        Methoxypolyethylene                                                                        Sigma    34F-0266 300 g.  20                                     Glycol 350                                                                    Benzyl Alcohol                                                                             BDH      23797    15 g.   1                                      Diclofenac Sodium                                                                          Prosintex                                                                              9123012  45 g.   3                                      Sodium Hyaluronate                                                                         Skymart  HG 1004  37.5 g. 2.5                                    (MW 679,000)                                                                  Sterile Water                                                                              Baxter   AW45R6   1200 ml.                                       balance                                                                       ______________________________________                                    

Procedure

set up stirring apparatus using a 3 liter stainless steel beaker

add water, Methoxypolyethylene Glycol 350, and Benzyl Alcohol and stirfor 20 minutes to mix

add Diclofenac Sodium and stir for 30 minutes to dissolve

add Hyaluronate Sodium slowly and stir initially at a high speed, butavoid splashing

after addition, stir at a slower speed for 90 minutes; the slower speedreduces the formation of air bubbles

the result is a clear, transparent, viscous gel which is put into acontainer. Once again instructions are given for administration and ifapplicable measuring devices (to provide a premeasured dosage amount)accompany the container.

Still other formulations are:

    ______________________________________                                        Formulation 3                                                                 3% Diclofenac in 2.5% HA Gel                                                  Formula     Supplier LOT      Amount   Percent                                ______________________________________                                        Sterile Water                                                                             Baxter   AW45K6   1200 ml  --                                     Methoxypolyethylene                                                                       Sigma    34F-0266 300G (273 ml)                                                                          20%                                    Glycol 350                                                                    Benzyl Alcohol                                                                            BDH      23797    15G (14 ml)                                                                            1%                                     Diclofenac Sodium                                                                         Prosintex                                                                              9123012  45 g     3%                                     Sodium Hyaluronate                                                                        Skymart  HG 1004  37.5 g   2.5%                                   MW 679,000                                                                    ______________________________________                                    

Procedure

Set up stirring apparatus using a 2 liter stainless steel beaker,

Add water, Methoxypolyethylene Glycol 350, and Benzyl Alcohol and stirfor 20 minutes to mix,

Add Diclofenoc Sodium and stir for 30 minutes to disolve,

Add Hyularonate Sodium slowly and stir initially at a high speed, butavoid splashing,

After addition, stir at a slower speed for 90 minutes, the slower speedreduces the formation of air bubbles,

The result is a clear transparent, viscous gel which is poured into jarsand tubes. Once again instructions accompany the container and whereapplicable appropriate devices for providing a premeasured amount of thecomposition accompany the container.

    ______________________________________                                        Formulation 4                                                                 5% IBUPROFEN IN 3.0% HA GEL 50 ml JAR                                         Formula    Supplier  LOT       Amount Percent                                 ______________________________________                                        Sterile Water                                                                            Baxter    AW45R6    196 ml --                                      Maglumine  Falk      15684     11 g   5.5%                                    Ibuprofen  DBH       19/241    10 g   5%                                      Benzy Alcohol                                                                            BDH       23797     2 g    1%                                      Glycerin   BDH       2579      2 g    1%                                      Hyaluronate                                                                   Sodium     Skymart   HG 1003   6 g    3%                                      Mol Wt 661,600                                                                ______________________________________                                    

PROCEDURE

Set up stirring apparatus using a 300 ml stainless steel beaker,

Add Sterile Water and Meglumine, and stir for 10 minutes,

Add Ibuprofen and stir for 15 minutes,

Add Benzyl Alcohol, followed by Glycerin and stir for 15 minutes,

Finally, add Hyaluronate Sodium slowly and stir initially at a hightorque to mix, but avoid splashing,

As the gel thickens, stir at a slow speed for 90 minutes.

    ______________________________________                                        Formulation 5                                                                 2% PIROXICAM IN 2.5% HA GEL                                                   Formula     Supplier  LOT       Amount Percent                                ______________________________________                                        Sterile Water                                                                             Baxter    AW45R6    200 ml --                                     Meglumine   Falk      15684     8 g    4%                                     Piroxicam   AMSA      1-010     4 g    2%                                     Hyaluronate Sodium                                                                        Skymart   HG 1003   5 g    2.5%                                   MW 661,600                                                                    ______________________________________                                    

PROCEDURE

Set up stirring apparatus using a 300 ml stainless steel beaker,

Add 200 ml of sterile water,

Add 8 grams of Meglumine and dissolve,

Very slowly add 4 grams of Piroxicam and stir for 20 minutes,

Slowly add 5 grams of Hyaluronate Sodium and stir at high speed,

Stir for 90 minutes at a slower speed

COMMENTS

A clear yellowish transparent gel

    ______________________________________                                        Formulation 6                                                                 5% IBUPROFEN CREAM, 50 ml JAR                                                                                         Per-                                  Formula    Supplier LOT        Amount   cent                                  ______________________________________                                        OILY PHASE                                                                    Liquid was DICDD                                                                         Brooks   L-1424     450 g    15%                                   Brookswax D                                                                              Brooks   P-490      480 g    16%                                   Glycerin   BDH      109109/2578                                                                              150 g(119 ml)                                                                          5%                                    AQUEOUS PHASE                                                                 Sterile Water                                                                            Baxter   AW45F1     1950 ml  --                                    Meglumine  Falk     15684      150 g    5%                                    Ibuprofen  BKH      19/241     150 g    5%                                    MW 200,00                                                                     Sodium Hyaluronate                                                                       Skymart  001        45 g     1.5%                                  Presevative Suttocide A Sutton                                                                SH-107     9 g        0.3%                                    ______________________________________                                    

PROCEDURE

A--Add all the ingredients of the oily phase A into a 4 liter stainlesssteel beaker, melt at 55° C., finally heat to 75% when Aqueous Phase Bis ready

B--Into a 3 liter stainless steel beaker, add 1950 ml water, set up, thestirring apparatus, add the Meglumine, stir to dissolve for 10 minutes,

Slowly add Ibuprofen, stir to dissolve for 20 minutes,

Very slowly add Sodium Hyaluronate and stir for one hour to dissolve allthe Sodium Hyaluronate,

Finally, heat to 75° C., with stirring for a total time of 30 minutes.

POUR B INTO A, both at a temperature of 75° C., slowly

Remove the heat source and stir with a strong vortex for one hour,

When the temperature has cooled down to 45° C. add preservativeSuttocide A,

Continue stirring at a slower speed until the temperature is 35° C.,

At 35° C. remove the propeller, pour into 50 ml jars.

    ______________________________________                                        Formulation 7                                                                 1% DICLOFENAC IN 3% HA Gel, 50 ml jar                                         Quantity 3000 ml                                                              Formula     Supplier LOT      Amount   Percent                                ______________________________________                                        Sterile Water                                                                             Baxter   AW45R6   2796 ml  --%                                    Glycerin    BDH      2579     50 g (71 ml)                                                                           3%                                     Benzyl Alcohol                                                                            BDH      23797    45 g (43 ml)                                                                           1.5%                                   Liquid wax DICDD                                                                          Brooks   191-175  90 g     3%                                     Diclofenac Sodium                                                                         Prosintex                                                                              9113003  30 g     1%                                     Hyaluronate Sodium                                                                        Skymout  HG 1004  90 g     3%                                     MW 679,000                                                                    ______________________________________                                    

PROCEDURE

Set up stirring apparatus using a 4 liter stainless steel beaker.

Add water, Glycerin, Benzyl Alcohol and Liquid wax DICDD and stir to mixthoroughly for 10 minutes

Add Diclofenac Sodium and stir for 30 minutes to dissolve.

Slowly add Hyaluronate Sodium, stirring at a high torque initiallyduring addition.

After addition stir at a slower speed for 90 minutes.

A white opaque viscous gel is formed.

    ______________________________________                                        Formulation 8                                                                 1% DICLOFENAC IN 3.0% HA Gel, 50 ml tube                                      Quantity 1500 ml                                                              Formula    Supplier LOT      Amount    Percent                                ______________________________________                                        Sterile Water                                                                            Baxter   AW45F1   1397 ml   --%                                    Glycerin   Life     1043     45 g (36 ml)                                                                            3%                                     Benzyl Alcohol                                                                           Caledon  02157    22.5 g (22 ml)                                                                          1.5%                                   Liquid wax DICDD                                                                         Brooks   191-175  45 g      3%                                     Diclofenac Sodium                                                                        Prosintex                                                                              9113003  15 g      1%                                     Sodium Hyaluronate                                                                       Skymart  HG 1003  45 g      3%                                     Mol. Wt. 661,600                                                              ______________________________________                                    

PROCEDURE

Set up stirring apparatus using a 3 liter stainless steel beaker.

Add water, Glycerin, Benzyl Alcohol and Liquiwax DICDD, stir to mix for10 minutes.

Add Diclofenac Sodium and stir for 30 minutes to dissolve.

Add Sodium Hyaluronate and stir for 90 minutes.

    ______________________________________                                        Formulation 9                                                                 HYANALGESE CREAM (L)                                                          50 ml tube                                                                    Quantity 3000 ml                                                                                                     PER-                                   FORMULA    SUPPLIER   LOT      AMOUNT  CENT                                   ______________________________________                                        A. Oily Phase                                                                 Liquid Wax DICDD                                                                         Brooks/Amisol       450 g   15.0%                                  Brookswax D                                                                              Brooks/Amisol       480 g   16.0%                                  Glycerine  Amisol              150 g   5.0%                                   B. Aqueous Phase                                                              Sterile Water                                                                            Baxter     AW4YA8   1950 ml --%                                    Maglumine  Falk                150 g   5.0%                                   Sodium Hyaluronate                                                                       Skymart    P01      45 g    1.5%                                   MW 207,000                                                                    Ibuprofen  BDH                 150 g   5.0%                                   Suttocide A                                                                              Sutton              9.0 g   0.3%                                   ______________________________________                                    

PROCEDURE

A.--Add all the ingredients of the oily phase into a 4 liter stainlesssteel beaker, melt at 55° C., finally heat to 75° C. when aqueous phaseis ready (at 75° C.) to pour in.

B.--Into another 4 liter stainless steel beaker, add 1950 ml water.

Set up the stirring apparatus and add the Meglumine

Stir to dissolve with high torque, then slowly add Ibuprofen

When the Ibuprofen is dissolved, slowly add Sodium Hyaluronate

Stir cold for one hour to dissolve all the ingredients

Finally heat to 75° C. and stir thoroughly throughout a 30 minute period

MIX B INTO A

Slowly pour B into A (both at 75° C.) with stirring

Immediately remove the hot plate (heat) and stir

Stir with a strong vortex for one hour

When the temperature is 45° C., add the preservative Suttocide A

Stir for about an hour to cool to 35° C.

At 35° C. remove the propeller and pour into 50 ml tubes

Pour 50 grams of the cream into each tube

    ______________________________________                                        1% BANAMINE IN 2.5% HA GEL                                                    (L) XPBN 041 Quantity 3000 ml                                                                                        PER-                                   FORMULA    SUPPLIER  LOT       AMOUNT  CENT                                   ______________________________________                                        Sterile Water                                                                            Baxter    AW4SA2    2400 m  1--%                                   Sodium Hyaluronite                                                                       Skymart   HE1003    75 g    2.5%                                   MW 661,600                                                                    *Banamine, Scheing   0 CNXB13  300 ml  1%                                     100 ml vial                                                                   Banamine,  Scheing   0 CNXB12  300 ml  1%                                     100 ml vial                    3000 ml                                        (50 mg/ml) 600                                                                        = 30,000 mg                                                                   = 30 grams Flunixin in 600 ml                                         ______________________________________                                         *Banamine contains Flunixin Meglumine (50 mg Flunixin per ml) or 83 mg        Flunixin Meglumine                                                       

PROCEDURE

Set up stirring apparatus using a 4 liter stainless steel beaker

Add water, stir with a strong vortex, then add sodium Hyoluronate slowly

Then immediately add the Banamine, stir the mixture for 4 hours.

One form of hyaluronic acid and/or salts thereof (for example sodiumsalt) and homologues, analogues, derivatives, complexes, esters,fragments, and sub-units of hyaluronic acid, preferably hyaluronic acidand salts and thereof, suitable for use with Applicant's invention is afraction supplied by Hyal Pharmaceuticals Limited. One such fraction isa 15 ml vial of Sodium hyaluronate 20 mg/ml (300 mg/vial--Lot 2F3). Thesodium hyaluronate fraction is a 2% solution with a mean averagemolecular weight of about 225,000. The fraction also contains water q.s.which is triple distilled and sterile in accordance with the U.S.P. forinjection formulations. The vials of hyaluronic acid and/or saltsthereof may be carried in a Type 1 borosilicate glass vial closed by abutyl stopper which does not react with the contents of the vial.

The fraction of hyaluronic acid and/or salts thereof (for example sodiumsalt) and homologues, analogues, derivatives, complexes, esters,fragments, and sub-units of hyaluronic acid, preferably hyaluronic acidand salts thereof, may comprise hyaluronic acid and/or salts thereofhaving the following characteristics:

a purified, substantially pyrogen-free fraction of hyaluronic acidobtained from a natural source having at least one characteristicselected from the group (and preferably all characteristics) consistingof the following:

i) a molecular weight within the range of 150,000-225,000;

ii) less than about 1.25% sulphated mucopolysaccharides on a totalweight basis;

iii) less than about 0.6% protein on a total weight basis;

iv) less than about 150 ppm iron on a total weight basis;

v) less than about 15 ppm lead on a total weight basis;

vi) less than 0.0025% glucosamine;

vii) less than 0.025% glucuronic acid;

viii) less than 0.025% N-acetylglucosamine;

ix) less than 0.0025% amino acids;

x) a UV extinction coefficient at 257 nm of less than about 0.275;

xi) a UV extinction coefficient at 280 nm of less than about 0.25; and

xii) a pH within the range of 7.3-7.9.

Preferably, the hyaluronic acid is mixed with water and the fraction ofhyaluronic acid has a mean average molecular weight within the range of150,000-225,000. More preferably, the fraction of hyaluronic acidcomprises at least one characteristic selected from the group (andpreferably all characteristics) consisting of the followingcharacteristics:

i) less than about 1% sulphated mucopolysaccharides on a total weightbasis;

ii) less than about 0.4% protein on a total weight basis;

iii) less than about 100 ppm iron on a total weight basis;

iv) less than about 10 ppm lead on a total weight basis;

v) less than 0.00166% glucosamine;

vi) less than 0.0166% glucuronic acid;

vii) less than 0.0166% N-acetylglucosamine;

viii) less than 0.00166% amino acids;

x) a UV extinction coefficient at 257 nm of less than about 0.23;

xi) a UV extinction coefficient at 280 nm of less than 0.19; and

xii) a pH within the range of 7.5-7.7

Applicants also propose to use sodium hyaluronate produced and suppliedby LifeCore™ Biomedical, Inc., having the following specifications:

    ______________________________________                                        Characteristics     Specification                                             ______________________________________                                        Appearance          White to cream                                                                colored particles                                         Odor                No perceptible odor                                       Viscosity Average   <750,000 Daltons                                          Molecular Weight                                                              UV/Vis Scan, 190-820 nm                                                                           Matches reference scan                                    OD, 260 nm          <0.25 OD units                                            Hyaluronidase Sensitivity                                                                         Positive response                                         IR Scan             Matches reference                                         pH, 10 mg/g solution                                                                              6.2-7.8                                                   Water               8% maximum                                                Protein              <0.3 mcg/mg NaHy                                         Acetate             <10.0 mcg/mg NaHy                                         ______________________________________                                        Heavy Metals, maximum ppm                                                     As    Cd    Cr     Co   Cu     Fe   Pb    Hg   Ni                             ______________________________________                                        2.0   5.0   5.0    10.0 10.0   25.0 10.0  10.0 5.0                            ______________________________________                                        Microbial Bioburden None observed                                             Endotoxin           <0.07 EU/mg NaHy                                          Biological Safety Testing                                                                         Passes Rabbit Ocular                                                          Toxicity Test                                             ______________________________________                                    

Another form of sodium hyaluronate is sold under the name HyaluronanHA-M5070 by Skymart Enterprises, Inc. having the followingspecifications:

    ______________________________________                                        Specifications' Test                                                          Results                                                                       ______________________________________                                        Lot No.           HG1004                                                      pH                 6.12                                                       Condroitin Sulfate                                                                              not detected                                                Protein            0.05%                                                      Heavy Metals      Not more than 20 ppm                                        Arsenic           Not more than 2 ppm                                         Loss on Drying     2.07%                                                      Residue on Ignition                                                                              16.69%                                                     Intrinsic Viscosity                                                                              12.75 dl/s (XW: 679,000)                                   Nitrogen           3.14%                                                      Assay             104.1%                                                      Microbiological Counts                                                                           80/g                                                       E. coli           Negative                                                    Mold and Yeast    Not more than 50/g                                          ______________________________________                                    

Other forms of hyaluronic acid and/or its salts, and homologues,derivatives, complexes, esters, fragments and sub units of hyaluronicacid may be chosen from other suppliers, for example those described inprior art documents provided the form of hyaluronic acid chosen issuitable for transport of the medicine.

The following references teach hyaluronic acid, sources thereof, andprocesses for the manufacture and recovery thereof which may besuitable.

U.S. Pat. No. 4,141,973 teaches hyaluronic acid fractions (includingsodium salts) having:

"(a) an average molecular weight greater than about 750,000, preferablygreater than about 1,200,000--that is, a limiting viscosity numbergreater than about 1400 cm³ /g., and preferably greater than about 2000cm³ /g.;

(b) a protein content of less than 0.5% by weight;

(c) ultraviolet light absorbance of a 1% solution of sodium hyaluronateof less than 3.0 at 257 nanometers wavelength and less than 2.0 at 280nanometers wavelength;

(d) a kinematic viscosity of a 1% solution of sodium hyaluronate inphysiological buffer greater than about 1000 centistokes, preferablygreater than 10,000 centistokes;

(e) a molar optical rotation of a 0.1-0.2% sodium hyaluronate solutionin physiological buffer of less than -11×10³ degree--cm² /mole (ofdisaccharide) measured at 220 nanometers;

(f) no significant cellular infiltration of the vitreous and anteriorchamber, no flare in the aqueous humour, no haze or flare in thevitreous, and no pathological changes to the cornea, lens, iris, retina,and choroid of the owl monkey eye when one milliliter of a 1% solutionof sodium hyaluronate dissolved in physiological buffer is implanted inthe vitreous replacing approximately one-half the existing liquidvitreous, said HUA being

(g) sterile and pyrogen free and

(h) non-antigenic."

Canadian Letters Patent 1,205,031 (which refers to U.S. Pat. No.4,141,973 as prior art) refers to hyaluronic acid fractions havingaverage molecular weights of from 50,000 to 100,000; 250,000 to 350,000;and 500,000 to 730,000 and discusses processes of their manufacture.

In order to determine the blood levels in patients using formulationsmade according to embodiments of the invention, a study of thepharmacokinetic profiles of two topical diclofenac formulations afterrepeat dosing were undertaken.

One such product was the product Voltarol Emulgel marketed in the UnitedKingdom by Geigy. The other was a Diclofenac preparation in HyaluronicAcid.

This was an open, repeat dose, crossover comparison using a randomizedbalanced block in six healthy volunteers.

The study consisted of administration with one, two week period inbetween periods, each period lasting fourteen days. The test articlesapplied were for the first six days of each period and the seventh daywas study day during which the final application is made and bloodsamples taken.

The approximate duration of the study including pre and post studyscreening was six weeks.

Doses

Diclofenac (3.0%) with Hyaluronic Acid (2.5%)

Dose: Approximately 2 g, three times daily

Route: Topical

(W1)Voltarol Emulgel, Diclofenac diethylammonium

salt 1.16 g aqueous gel (Geigy)

Dose: Approximately 2 g, three times daily

Route: Topical (W1)

ADMINISTRATION: to suitable patients

Subjects applied one of the designated test articles topically to thecalves and massaged into the skin, in a dose of approximately 2 g perapplication three times a day for six consecutive days. The size of a 2g dose was prepared by comparison with a silicone example given to eachsubject.

On the seventh day, the cream was applied once, in the same manner asbefore, under the supervision of the staff of the Clinical InvestigationUnit.

After a washout period of one week the procedure was repeated with thealternate test article.

The following were the results of the tests:

(H=hyaluronic acid formulation)

(V=Voltarol Emulgel)

    __________________________________________________________________________    All concentrations ng ml -1                                                   TIME POINT (hours)                                                            SUBJECT                                                                            0  0.25                                                                             0.5                                                                              1  2  3  4  5  6  8  10 12                                      __________________________________________________________________________    PERIOD 1                                                                      H-1  10.3                                                                             7.1                                                                              6.4                                                                              ND ND 5.4                                                                              6.5                                                                              5.1                                                                              ND ND ND ND                                      H-2  ND 5.1                                                                              ND 5.1                                                                              ND ND ND ND ND 5.1                                                                              ND ND                                      ND   ND ND 5.5                                                                              5.2                                                                              ND ND ND ND ND ND ND V-3                                     ND   ND ND ND ND ND ND ND ND ND ND ND H-4                                     ND   ND ND ND ND ND ND ND ND ND ND ND V-5                                     ND   ND ND ND ND ND ND 8.4                                                                              ND ND ND ND V-6                                     PERIOD II                                                                     V-1  ND ND ND ND ND ND ND ND ND ND ND ND                                      V-2  ND ND ND ND ND ND ND ND ND ND ND ND                                      H-3  ND ND ND ND ND ND ND ND ND ND ND ND                                      V-4  ND ND ND ND ND ND ND ND ND ND ND ND                                      H-5  ND ND ND ND ND ND ND ND ND ND ND ND                                      H-6  ND ND ND ND ND ND ND ND ND ND ND ND                                      __________________________________________________________________________     ND = NONE DEDECTED (>5.0 ng ml.sup.-1)                                   

Other tests were undertaken to determine blood levels comparing Proflex(a formulation containing Ibuprofen) and the following formulationcontaining hyaluronic acid and Ibuprofen.

    __________________________________________________________________________    HYANALGESE CREAM (L) X PB 022                                                 -50 ml tube                                                                   Quantity 3000 ml                                                              FORMULA   SUPPLIER                                                                              LOT   AMOUNT                                                                             PERCENT                                          __________________________________________________________________________    A. Oily Phase                                                                 Liquid Wax DICDD                                                                        Brooks/Amisol 450                                                                              g 15.0%                                            Brookswax D                                                                             Brooks/Amisol 480                                                                              g 16.0%                                            Glycerine Amisol        150                                                                              g 5.0%                                             B. Aqueous Phase                                                              Sterile Water                                                                           Baxter  AW4YA8                                                                              1950                                                                             ml                                                                              --%                                              Meglumine Falk          150                                                                              g 5.0%                                             Sodium Hyaluronate                                                                      Skymart PO1   45 g 1.5%                                             MW 207,000                                                                    Ibuprofen BDH           150                                                                              g 5.0%                                             Suttocide A                                                                             Sutton        9.0                                                                              g 0.3%                                             __________________________________________________________________________

The following were the results

    __________________________________________________________________________    SUBJECT                                                                            Time after administration (Hours)                                        Number                                                                             PD 0  0.25                                                                             0.5                                                                              1  2  3  4  5  6  8  10 12                                   __________________________________________________________________________    (A) PROFLEX                                                                   1    ND 0.41                                                                             0.37                                                                             0.37                                                                             0.32                                                                             0.30                                                                             0.27                                                                             0.27                                                                             0.24                                                                             0.37                                                                             0.31                                                                             0.31                                                                             0.16                                 2    ND 0.12                                                                             0.12                                                                             0.08                                                                             0.11                                                                             0.12                                                                             0.12                                                                             0.07                                                                             0.08                                                                             0.09                                                                             0.08                                                                             ND 0.06                                 3    ND 0.09                                                                             0.08                                                                             0.07                                                                             ND ND ND ND ND ND ND ND ND                                   4    ND 0.12                                                                             0.14                                                                             0.16                                                                             0.11                                                                             0.11                                                                             0.25                                                                             0.24                                                                             0.17                                                                             0.13                                                                             0.16                                                                             0.11                                                                             0.13                                 5    ND 0.14                                                                             0.19                                                                             0.19                                                                             0.15                                                                             0.16                                                                             0.16                                                                             0.14                                                                             0.12                                                                             0.11                                                                             0.13                                                                             0.10                                                                             0.07                                 6    ND 0.11                                                                             0.09                                                                             0.09                                                                             0.06                                                                             0.07                                                                             0.05                                                                             0.05                                                                             0.05                                                                             ND ND ND ND                                   Mean 0.00                                                                             0.17                                                                             0.17                                                                             0.16                                                                             0.13                                                                             0.13                                                                             0.14                                                                             0.13                                                                             0.11                                                                             0.12                                                                             0.11                                                                             0.09                                                                             0.07                                 S.D. 0.00                                                                             0.12                                                                             0.10                                                                             0.11                                                                             0.10                                                                             0.10                                                                             0.10                                                                             0.10                                                                             0.08                                                                             0.13                                                                             0.11                                                                             0.12                                                                             0.06                                 (B) HYALURONIC ACID AND IBUPROFEN                                             1    ND 0.11                                                                             0.11                                                                             0.12                                                                             0.08                                                                             0.08                                                                             0.09                                                                             0.11                                                                             0.12                                                                             0.08                                                                             0.11                                                                             0.16                                                                             0.14                                 2    ND 0.22                                                                             0.21                                                                             0.26                                                                             0.17                                                                             0.24                                                                             0.24                                                                             0.25                                                                             0.23                                                                             0.19                                                                             0.19                                                                             0.20                                                                             0.14                                 3    ND 0.17                                                                             0.10                                                                             0.12                                                                             0.09                                                                             0.08                                                                             0.07                                                                             0.06                                                                             ND 0.06                                                                             0.26                                                                             0.09                                                                             0.05                                 4    ND ND ND ND ND ND ND ND ND ND ND ND ND                                   5    ND 0.17                                                                             0.16                                                                             0.16                                                                             0.12                                                                             0.09                                                                             0.10                                                                             0.11                                                                             0.10                                                                             0.09                                                                             0.10                                                                             0.07                                                                             ND                                   6    ND 0.07                                                                             0.07                                                                             0.09                                                                             ND ND ND ND ND ND ND ND ND                                   Mean 0.00                                                                             0.12                                                                             0.11                                                                             0.13                                                                             0.08                                                                             0.08                                                                             0.06                                                                             0.09                                                                             0.08                                                                             0.07                                                                             0.11                                                                             0.09                                                                             0.06                                 S.D. 0.00                                                                             0.08                                                                             0.07                                                                             0.08                                                                             0.06                                                                             0.08                                                                             0.08                                                                             0.09                                                                             0.09                                                                             0.07                                                                             0.10                                                                             0.08                                                                             0.07                                 __________________________________________________________________________     ND None detected <0.05 μg/ml                                          

The above clearly indicates that the blood levels are much less usinghyaluronic acid to administer the NSAID.

PRELIMINARY REPORT

A trial was conducted using a gel composition (Number 109) comprising 3%Diclofenac in 2.5% Hylauronic Acid as previously described and acomposition containing Diclofenac sodium salt 3% but not including anyform of hyaluronic acid. (Number 112) The trial was conducted with 60patients who were randomly assigned to test preparations number 109 or112. The trial has not been completed as yet but so far 31 patients havefinished the protocol. Patients were diagnosed:

4 Rheumatoid arthritis of the knee

8 Myofascial trigger points in the M. trapezius area

12 Periarthropathies of knee without effusion

7 Periarthropathies with effusion in the knee joint

The 31 patients were aged 22-75 years (27 females, 4 males). Allpatients were hospitalized. Patients entering the trial were thoroughlyexamined and type of extraarticular or articular rheumatism assessed.

On day 1 baseline pain was assessed on the 10 cm visual analogue scale(VAS) and pain measurement of the quantititative pain sensitivity usinga pressure tolerance meter (PTM) were performed. Then testgel--approximately 2.g--was massaged on to the skin of maximum pain.Gels were applied 3 times daily.

0.5, 1, 1.5, and 2 hours after morning application measurements of painsensitivity were carried out and values recorded.

This procedure was countinued on day 2, 3 and 4; measurements (VAS andPTM) of pain severity were done on day 1, 2 and 4.

Prior of the beginning of the study and at the end on day 4, physician'sglobal assessment, assessment of swelling, tenderness and limitation ofmovement were recorded.

As the study is ongoing statistical evaluation is not yet available. Forfurther details see Table 1.

                  TABLE 1                                                         ______________________________________                                                       Composition                                                                             Composition                                          ______________________________________                                        Reaction         109, n = 16 112, n - 15                                      Good Alleviation  13          8                                               of pain                                                                       Moderate Alleviation                                                                            2           2                                               of pain                                                                       No Alleviation    1           5                                               of pain                                                                       ______________________________________                                    

From the data recorded we have concluded that the patients to whomcomposition 109 was administered did better in terms of earlier andlonger lasting analgesic effect (up to 4 hours) than the 112 compositionespecially in patients with myofascial trigger points and withperiarthropathies of the knee joints without effusions. Neithercomposition 109 nor composition 112 treated patients showed any effecton swelling if any swelling exist at all. Systemic side effects have notbeen observed; one patient to whom composition 112 was administeredshowed reddening of the skin on the site of application.

Any intake of system NSAIDS, corticosteroids and other analgesics wasnot allowed one week before and during the trial.

EXAMPLES

The following examples are offered to illustrate uses of Applicants'invention.

Example 1

A male patient had a number of lesions (basal cell carcinoma), includingone on his forehead which was a combination of major "horny epithelium"and some degree of ulceration. After continuous treatment withFormulation 1 (several times per day for several weeks of dosage amountssqueezed from tubes as ribbons of composition), the lesions showedepithelialization, no hemorrhagic areas, and no initiated areas (as theywere in the past without our treatment). The "horny epithelium" andulceration of the forehead lesion were also gone. The patient had acomplete successful response with the formulation. All basal cellcarcinoma lesions had been resolved and disappeared. There has been norecurrence.

Example 2

60 year old male tennis player had sore elbow and basal cell carcinomaon forearm proximate sore elbow. Patient tried Formulation 1 to abatepain in tennis elbow. (Dr. Falk was not treating this patient foranything at the time, did not know of the basal cell proximate the elbowand merely offered the formulation for pain relief of the elbowinstucting the patient to squeeze a ribbon of the composition and applyand rub into the sore elbow). However, the formulation "spilled" overonto the Patient's basal cell carcinoma. Patient was planning to havebasal cell carcinoma removed surgically by another doctor, but when thepatient returned to see the doctor, the basal cell carcinoma wasdisappearing (because of spill-over of Formulation 1). Dr. Falk was thenadvised and treatment was now undertaken by Dr. Falk with directapplication of Formulation 1 to the lesion 3 times a day for twoadditional weeks. After two weeks, the basal cell carcinoma disappeared.There has been no recurrence.

Example 3

Male, mid to late 40's had severe basal cell carcinoma on left temple.Doctors recommended its removal by surgery. However, the surgery wouldhave been risky because of the lesion's proximity to facial nerves.

Patient saw Dr. Falk who gave him Formulation 2 to be applied in dosageamounts 3 times daily.

After 14 days, 75% of the lesion was gone. Surgery was postponed and thetreatment was continued. Application of dosage amounts of Formulation 2was continued for an additional two weeks. At the end of the 2-weekperiod, the lesion was completely resolved and disappeared without anysurgery being required. There has been no recurrence.

Example 4

Male, early 40's, had recurrent actinic keratoses lesion on his righttemple. Early attempts at removal by third parties involved theapplication of liquid nitrogen (twice) without final resolution. Thelesion kept recurring. The patient was sent to Dr. Falk who treated thelesion with Formulation 1 with applications of dosage amounts 3 timesdaily for 7 days. After 7 days, the lesion was completely resolved withno subsequent recurrence.

Example 5

A male patient suffering from kyphosis suffered from constant back pain.Taking analgesics orally and rubbing back preparations onto his back didlittle to alleviate the back pain. When NSAIDs in hyaluronic acid(sodium hyaluronate) were applied directly to the back, the back paineased and disappeared.

With indomethacin (dissolved in N-methyl glucamine) and naproxen bothdissolved in hyaluronic acid, the patient experienced some side effects.However, with Toradol™ (the +/-! form tromethamine salt of ketorolac--aprostaglandin biosynthesis inhibitor and analgesic andanti-inflammatory, the back pain eased and disappeared for some time andthere were no side effects. The compositions were applied generouslyonto the sites of back pain.

Example 6

A male patient with basal cell carcinoma was first treated by anoncologist who attempted to surgically excise the lesion (withoutsuccess) and then irradiated the lesion again without success. Thepatient then attended before Dr. Falk who applied Applicant'sformulation (diclofenac with sodium hyaluronate and excipients).Application was made three times daily for about a month and the lesiondisappeared. Some excoriation anterior and slightly superior developedover the last two weeks but was cleared by the application of hyaluronicacid by itself.

This resolution clearly indicates that even with prior applications ofunsuccessful therapies (surgery and irradiation), Applicant'sformulations can be used successfully.

Example 7

In another patient, a drug (methotrexate) was carried in hyaluronic acidand applied topically to a patient with psoriasis. The formulation wasabsorbed and the psoriasis cleared.

Example 8

A patient with dermal (skin) metastases in a fibratic scar form andmetastatic cancer in the form of musculoskeletal involvement in herthorax.

On topical application of our formulation comprising diclofenac(Voltaren) in hyaluronic acid (sodium hyaluronate), her pain decreaseddramatically and her skin and boney involvements steadily improved.

TOPICAL DICLOFENAC ACID 3% IN HYALURONIC ACID GEL (2.5%) BASE

A practitioner reviewed the effectiveness of topical Diclofenac Acid 3%in hyaluronic acid gel (2.5%) base in acute traumatic injuries of nolonger than 3 days duration. The cases were all in the spectrum of agesbetween 18 and 65. Normal exclusion criteria were followed regardingexclusion of pregnancy, aspirin or N.S.A.I.D., allergies or activepeptic ulceration.

As an overall, the following impressions were gained from 30 cases:

1. The topical H.D. (composition comprising sodium hyaluronate anddiclofenac) had an obvious analgesic action with onset occurring rapidlywithin one hour; this is a phenomenon not obviously seen with othernon-steroidals that we have used.

2. There was a very definite patient acceptance of the gel as a form oftreatment, being logical, easy to apply, without local or systemic sideeffects, rapid absorption with no staining of clothing.

3. The anti-inflammatory action was equivalent on a "guestimate" basedon experience of similar injuries to oral N.S.A.I.D.s, without thethreat or risk of side effects.

In summary, compared with other topical N.S.A.I.D.s the analgesic effectis distinct, the anti-inflammatory is equal to oral N.S.A.I.D.s and thepatients' acceptance is far superior to any other diclofenac orpiroxicam topical that the practitioner evaluated.

Following the practitioner's basic preamble regarding the parallelism oftopical N.S.A.I.D.s and topical steroids, the practitioner has used theformer in contact dermatitis, insect bites and U.V. erothema, all withvery positive effects, again pointing direction to trials of a doubleblind nature in these fields.

    __________________________________________________________________________    CHRONIC CONDITIONS - EVALUATIONS                                                                               Positive (P)                                 Patients                                                                           Date                        Negative (N)                                 Initials                                                                           of   File                   Unable to                                    (M) or (F)                                                                         Birth                                                                              No.                                                                              Diagnosis                                                                           Comments on Outcome                                                                         Comment (U)                                  __________________________________________________________________________    2.5% HYALURONIC ACID WITH 3% DICLOFENIC ACID (HD)                             LA (M)                                                                             11.04.56                                                                              Hyper-                                                                              Severe discomfort following                                                                 P                                                         aesthesia                                                                           extensive surgery to dorsal                                                   spine with insertion of                                                                     Example of                                                      rods in 1989. Even contact                                                                  peripheral                                                      with clothes produced sig-                                                                  action on                                                       nificant discomfort.                                                                        super-                                                          Initially treated with EMLA                                                                 sensitiza-                                                      with only transient anaes-                                                                  tion of                                                         thetic results, however                                                                     nerve                                                           even after 3 days treatment                                                                 ending                                                          with Hyal diclofenac acid                                                                   queried.                                                        noticed marked decrease in                                                    supersensitivity which has                                                    continued for at least 4                                                      weeks while still using gel.                               KB (F)                                                                             08.06.58                                                                              Chronic                                                                             Treated right knee which was                                                                P                                                         chondro-                                                                            worse initially and was                                                 malacia                                                                             amazed at the response, then                                            perhaps                                                                             started to treat left knee                                              dating back                                                                         that was not so painful,                                                to 1976.                                                                            again with positive response.                                                 Here we have a built-in con-                                                  trol.                                                      DB (F)       Chronic                                                                             Initially felt some improve-                                                                N                                                         neurogenic                                                                          ment which was not continued                                            pain in                                                                             although initially quite                                                ankle with                                                                          positive - query placebo                                                associated                                                                          reaction.                                                               dysaesthesia.                                                    DC (F)                                                                             07.11.51                                                                              Chronic back        N                                                         pain - query                                                                  due to facet                                                                  syndrome or                                                                   trigger                                                                       points,                                                                       really diag-                                                                  nosis                                                                         uncertain.                                                       HYALURONIC ACID WITH 3% DICLOFENAC ACID (HD)                                  CC   18.01.25                                                                              Chronic cap-                                                                        Definite effect over knee                                                                   P                                                         sulitis                                                                             where application to tar-                                               right hip                                                                           get distance short. No                                                  right knee                                                                          obvious effect over hip.                                   AG (F)                                                                             07.11.58                                                                              Myositis in                                                                         Initially given placebo in                                                                  P                                                         rhomboids                                                                           error, only marginal or                                                 muscles                                                                             minimal effect, if any.                                                 following                                                                           Found active to be effect-                                              motor tive while being used, did                                              vehicle                                                                             not cure condition which                                                accident                                                                            needed trigger point                                                          therapy.                                                   CH (F)                                                                             22.08.61                                                                              Chronic                                                                             No significant effect, nor                                                                  N                                                         relapsing                                                                           has aggressive therapy since                                            tendonitis                                                                          including injection with                                                right elbow                                                                         cortisone and numerous                                                        opinions.                                                  SH (F)                                                                             16.07.55                                                                              Tendonitis                                                                          Control of tendonitis while                                                                 P                                                         and myositis                                                                        using preparation. Is now                                                     back at work.                                              DM (M)                                                                             17.06.47                                                                              Neuronitis                                                                          This patient has a very                                                                     U                                                               unusual pain in his left                                                      groin following nerve                                                         injury, with the use of                                                       preparation noticed de-                                                       crease in pain sensation                                                      while on medication.                                                          Hyperaesthesia altered                                                        although pain (which may be                                                   phantom) still present.                                    PJ   15.06.45                                                                              Capsulitis                                                                          Symptoms improved 50% while                                                                 U                                                         of right                                                                            using Hyal diclofenac acid,                                             wrist however, on discontinuation                                                   pain reappeared. Exact                                                        etiology uncertain.                                        DJ (F)       Dorsal                                                                              Control while using gel                                                                     P                                                         myositis                                                                            equal and with less side                                                      effects than tiger balm.                                                      Controlled symytoms while                                                     using medication. Exact                                                       diagnosis as to cause of                                                      myositis uncertain.                                        DK (F)                                                                             27.08.38                                                                              Severe                                                                              This patient has had capsu-                                                                 P                                                         capsulitis                                                                          litis left shoulder for many                                            left  years and treated with only                                                                 Extremely                                                 shoulder                                                                            transient relief with corti-                                                                rewarding                                                       sone injections, poor relief                                                                case                                                            with topical piroxicam. Was                                                   started on topical diclo-                                                     fenac acid and noticed                                                        relief of pain in 20 minutes                                                  continuing for 4-6 hours.                                                     See letter March 11/92. At                                                    present is using H.D. regu-                                                   larly, has found it to be                                                     useful in other areas of                                                      chronic pain. Is President                                                    North American Chronic                                                        Pain Association, has good                                                    insight into medication and                                                   placebos etc. Has two D.C.S.                                                  implants.                                                  JL (M)                                                                             10.12.45                                                                              Chronic                                                                             Pain has failed to respond                                                                  N                                                         myositis                                                                            to many aggressive treat-                                               secondary to                                                                        ments.                                                                  query facet                                                                   syndrome                                                         RMC (F)                                                                            13.06.57                                                                              Neuronitis                                                                          It is a difficult case with                                                                 U                                                         following                                                                           considerable overlay, she                                               facet obtained some relief with                                               rhizotomy                                                                           H.D., would estimate 30-40%                                             with result-                                                                        Interestingly hyper-                                                    ing pain in                                                                         anaesthesia was decreased.                                              her back                                                         RM (F)                                                                             20.08.52                                                                              Chronic                                                                             Using H.D. significant                                                                      P                                                         capsulitis                                                                          improvement in pain while                                                     used, on stopping treatment                                                   recurrence of pain, needed                                                    intra-articular cortisone.                                 GM (F)       Sub-acute                                                                           Rapid resolution of pain                                                                    P                                                         tendonitis                                                                          within one day and positive                                             right ankle                                                                         return of function.                                        PM (F)                                                                             20.09.46                                                                              Acute on                                                                            Rapid analgesic response                                                                    P                                                         chronic                                                                             with rapid settlement.                                                  osteo-                                                                        arthritis of                                                                  first meta-                                                                   tarsal                                                                        phalyngeal                                                                    joints                                                           DN (F)                                                                             10.03.44                                                                              Chronic                                                                             Excellent response to appli-                                                                P                                                         fasciaitis                                                                          cation of H.D. with occlu-                                              of feet                                                                             sion. Had failed to respond                                                   to oral N.S.A.I.D.s and                                                       physiotherapy. Query posi-                                                    tive result due to short                                                      application target distance                                                   in a vascular tissue.                                      BP (F)                                                                             04.03.20                                                                              Severe                                                                              Initially one knee treated                                                                  P                                                         chronic                                                                             with such good results                                                  arthritis of                                                                        both knees treated, see                                                                     Side                                                      the knee.                                                                           letter. Not only did pain                                                                   effects-                                                  Unable to                                                                           decrease but marked swell-                                                                  non/Inci-                                                 take oral                                                                           ing around knees. Signifi-                                                                  dental                                                    N.S.A.I.D.s                                                                         cant relief of pain and                                                                     resolution                                                      increase in movement as a                                                                   of area of                                                      result of this and perhaps                                                                  thrombo-                                                        reduction of swelling.                                                                      phlebitis                                                       Interestingly has severe                                                                    below area                                                      superficial varicose veins,                                                                 of treat                                                        developed thrombophlebitis                                                                  ment                                                            around right knee and the                                                     area treated by chance                                                        showed far less redness and                                                   tenderness than the throm-                                                    bophlebitis below this area.                               SP (M)                                                                             06.11.48                                                                              Idio- Has had similar episodes                                                                    U                                                         pathic                                                                              with poor response to mny                                               diffuse                                                                             treatments including                                                    capsulitis                                                                          N.S.A.I.D.s per os                                                      of hands                                                         WS   04.06.45                                                                              Chronic                                                                             Has been exposed to numer-                                                                  U                                                         neuronitis                                                                          ous treatments including                                                due to                                                                              tow attempts of surgery                                                 injury to                                                                           without effect. There is                                                lateral                                                                             decrease in hyperaesthesia                                              cutaneous                                                                           but no change in pain.                                                  nerve of                                                                      thigh                                                            MS (F)                                                                             04.06.28                                                                              Chronic                                                                             Failed to respond to number                                                                 P                                                         capsulitis                                                                          of treatments, good back-                                                     ground resolution of pain,                                                    however, still had acute                                                      pain with certain movements.                               IS (F)                                                                             15.01.48                                                                              Chronic                                                                             Had failed to respond to                                                                    P                                                         capsulitis                                                                          numerous treatments                                                           including oral and topical                                                    N.S.A.I.D.s Using H.D                                                         there was equivalent control                                                  of pain as with other                                                         therapies which lasted while                                                  medication was used.                                                          Referred for surgical                                                         opinion.                                                   GS (F)                                                                             26.03.47                                                                              Chronic                                                                             Oral diclofenac acid dis-                                                                   P                                                         tendo-                                                                              continued due to gastritis                                              sinovitis                                                                           and also history of ulcera-                                             tion. tion. Control using H.D.                                                      equal to or better than oral                                                  N.S.A.I.D.s.                                               VK (F)                                                                             01.01.39                                                                              Chronic                                                                             Good relief of pain and                                                                     P                                                         tendonitis                                                                          tenderness while using H.D.                                                                 for pain                                                        however on discontinuation                                                                  N                                                               of gel symptoms returned,                                                                   for                                                             treated with intramuscular                                                                  resolution                                                      steroids.                                                  GH (M)                                                                             03.11.21                                                                              Acute on                                                                            In view of age and general                                                                  P                                                         chronic                                                                             parous medical condition,                                                                   Commented                                                 osteo-                                                                              ideal for topical. Had                                                                      on better                                                 arthritis                                                                           been previously on topical                                                                  absorption                                                left hand                                                                           piroxicam for left shoulder                                                                 compared                                                        capsulitis.   to topical                                                                    piroxicam                                    JA (M)                                                                             06.02.58                                                                              Severe post-                                                                        Produced good superficial                                                                   P                                                         traumatic                                                                           analgesia especially                                                    and surgical                                                                        staples were irritating sub-                                            osteo-                                                                              cutaneous tissue, little                                                arthritis of                                                                        effect on deeper, severe                                                left leg                                                                            osteoarthritic pain of knee.                                            with staples.                                                                       This pain was of consider-                                              Poor result                                                                         able severity, needing nar-                                             to oral                                                                             cotics.                                                                 N.S.A.I.D.s                                                                   also gastric                                                                  irritation.                                                      IM (M)                                                                             30.11.51                                                                              Chronic                                                                             Severe rhomboid inflammation                                                                P                                                         superficial                                                                         right side, treated with                                                myositis                                                                            H.D., very definite improve-                                                  ment in pain and tenderness.                               TK (F)                                                                             23.04.70                                                                              Acute on                                                                            Excellent rapid analgesic                                                                   P                                                         chronic                                                                             followed by anti-inflammatory                                           capsulitis                                                                          response in young women who                                             due to                                                                              could not take oral N.S.A.D.s                                           sports                                                                              due to past gastritis.                                                  injury right                                                                  hand                                                             AD (F)                                                                             03.01.49                                                                              Chronic                                                                             Poor response to H.D. After                                                                 N                                                         diffuse pain                                                                        intensive investigation and                                             thought to                                                                          numerous consultations and                                              be myositis                                                                         treatment, pain still un-                                                     diagnosed and unresponsive.                                NH (F)                                                                             25.03.25                                                                              Subacute                                                                            Excellent response analgesic                                                                P                                                         capsulitis                                                                          and anti-inflammatory-wise                                              right ankle                                                                         within a few days. Marked                                                     clinical improvement. In                                                      view of this patient's                                                        parous general medical con-                                                   dition and hypertension, not                                                  suitable for oral NSAIDs.                                  MD   18.04.34                                                                              Subacute                                                                            Had failed to respond to                                                                    N                                                         rheumatoid                                                                          oral N.S.A.I.D.s, which                                                 arthritis                                                                           caused gastritis, tried                                                       topical piroxicam with nega-                                                  tive effects. Negative                                                        response to H.D.                                           MW (F)                                                                             07.05.46                                                                              Heberden's                                                                          Very slow positive outcome,                                                                 P                                                         nodes, pain-                                                                        initially improvement in                                                ful, swollen                                                                        pain followed by reduction                                              causing                                                                             in swelling. Etiology of                                                difficulty                                                                          this condition is unknown,                                              in movement                                                                         partly genetic. Would have                                                    been interesting to treat                                                     alternate digits, plus or                                                     minus thermographic confir-                                                   mation.                                                    LP (F)                                                                             20.07.23                                                                              Acute on                                                                            Initially treated with                                                                      P                                                         sub-acute                                                                           Idarac, poor response over-                                             osteo-                                                                              all, some improvement in                                                arthritis of                                                                        generalised arthritis of                                                the hands                                                                           hands but none on Heberden's                                            with  nodes. Pain flared or stop-                                             Heberden's                                                                          ping Idarac due to gastritis.                                           nodes Started on H.D., especially                                                   favourable results with sub-                                                  sidence of tenderness of                                                      nodes and settling of                                                         arthritis. Interestingly                                                      enough, no flare up on dis-                                                   continuation after one month.                              JG (F)                                                                             24.11.50                                                                              Post facet                                                                          Had failed to respond to                                                                    U                                                         rhizotomy                                                                           oral N.S.A.I.D.s and                                                    hyper-                                                                              E.M.L.A. Application of                                                 aesthesia,                                                                          H.D. improved the surface                                               with marked                                                                         pain significantly but had                                              pain and                                                                            no effect on the deeper                                                 hyper-                                                                              pain. My impression was                                                 aesthesia                                                                           that the deeper pain was                                                between                                                                             due to section of the facet                                             scapulae                                                                            nerve and beyond the reach                                                    of the topical medication.                                                    There is little doubt that                                                    the skin sensitivity was                                                      decreased.                                                 SW (F)                                                                             10.09.39                                                                              Knee pain                                                                           Upset in past due to oral                                                                   P                                                         due to                                                                              N.S.A.I.D.s., also hyper-                                                                   (Effec-                                                   chondro                                                                             tension made one loathe to                                                                  tive while                                                malacia                                                                             use this medication with                                                                    being                                                           serum levels. Good anal-                                                                    used)                                                           gesic and anti-inflammatory                                                                 Condition                                                       action, however on discon-                                                                  only cured                                                      tinuation pain flared. Seen                                                                 by surgery                                                      for arthroscopic surgery                                                      with relief of pain.                                       __________________________________________________________________________     *Two types of painresponse in only one                                        1. Interestingly in the whole series, there was not one case of local sid     effects and as expected from past studies, no general or systemic. Since      this report was prepared we have had one case of mild folliculitis which      responded to discontinuation of treatment, will rechallenge.                  2. A number of patients commented that they felt the gel improved the         texture and softness of their skin, and commented that it was messy or        stained their clothes.                                                        3. In one case of topical thrombophlebitis where the inflamed vein crosse     the area of treatment, the vein in the area of treatment improved while       that outside at a distance did not. Again, similar to using oral              N.S.A.I.D.s. ***.                                                        

Photographs were taken of patients with basal cell carcinoma FIGS. 6-11photographs, and of mice with tumors induced in the skin of the hindlegs (FIG. 12 photographs). The patients were treated by usingcombinations of NSAIDS, (non-steroidal anti-inflammatory drugs) andhyaluronic acid (including sodium hyaluronate) according to theinvention (3% diclofenac in 2.5% sodium hyaluronate gel base). Each ofthe six sets of Figures made up of photographs of the different personsshould include a legend describing or explaining each picture asfollows:

Legend for FIGS. 6a and 6b should read: Patient: W. D., male, 82 yearsDiagnosis: Basal cell carcinoma Treatment: NSAIDS plus HA gel. 3 timesper day FIG. 6a: June, 1991 FIG. 6b: December, 1991

Legend for FIGS. 7a and 7b should read: Patient: M. F., male, 45 yearsDiagnosis: Basal cell carcinoma Treatment: NSAIDS plus HA gel. 3 timesper day FIG. 7a: January, 1992 FIG. 7b: April, 1992

Legend for FIGS. 8a, 8b, 8c and 8d should read: Patient: H. A., male, 82years Diagnosis: Basal cell carcinoma Treatment: NSAIDS plus HA gel. 3times per day FIG. 8a: Jan. 26, 1992 FIG. 8b: Mar. 16, 1992 FIG. 8c:Jan. 26, 1992 FIG. 8d: Mar. 16, 1992

Legend for FIGS. 9a, 9b, 9c and 9d should read: Patient: R. F., male, 64years Diagnosis: Basal cell carcinoma Treatment: NSAIDS plus HA gel. 3times per day FIG. 9a: Jan. 26, 1992 FIG. 9b: Mar. 16, 1992 FIG. 9c:Jan. 26, 1992 FIG. 9d: Mar. 16, 1992

Legend for FIGS. 10a, 10b, 10c and 10d should read: Patient: R. W.,male, 86 years Diagnosis: Basal cell carcinoma Treatment: NSAIDS plus HAgel. 3 times per day FIG. 10a: Jan. 26, 1992 FIG. 10b: Mar. 16, 1992FIG. 10c: Jan. 26, 1992 untreated FIG. 10d: Mar. 16, 1992 untreated

Legend for FIGS. 11a, 11b and 11c should read: Patient: E. D., female,70 years Diagnosis: Basal cell carcinoma Treatment: NSAIDS plus HA gel.3 times per day FIG. 11a: Apr. 20, 1992 FIG. 11b: May 13, 1992 FIG. 11c:Jul. 7, 1992

The Legend for FIG. 12 (FIGS. 12a and 12b) relate to: Mouse Strain: DBA₂Tumour: p815 FIG. 12a: control, 19 days FIG. 12b: Novantrone plus HA gel19 days

The mice shown in FIGS. 12a and 12b had tumours induced in the skin oftheir hind legs and dosage amounts (2 ml) of Novatrone (10 mg. perdosage amount) (MITOXANTRONE (t.m.) and 2.5% sodium hyaluronate wereapplied (rubbed onto) the skin at the site of the pathology. The tumoursreduced in size (See FIG. 12b) clearly illustrating the percutaneousdelivery of the medicine by the hyaluronic acid. (See FIG. 12).

The following additional comments are made with respect to the patients.

With respect to R. W. and FIG. 10, the reader will note in FIGS. 10a and10c the patient suffered from basal cell carcinoma on his back (FIG.10c) and his temple (FIG. 10a). Because of the age of the individual(86) the basal cell carcinoma on his back could not be reached by himfor application of the medication. Thus the basal cell carcinoma in 10cremained untreated and grew (see FIG. 10d). However, the portionindicated in 10a on his temple could be reached and after application ofthe basal cell carcinoma formulation to the temple and forehead theresults are as in 10b; the basal cell carcinoma is disappearing. Thus,the gentlemen's own method of treatment acted as a control.

With respect to R. F. and FIG. 9, two areas of basal cell carcinoma inneed of treatment are shown by the arrows in FIGS. 9a and 9c and theresults are shown in FIGS. 9b and 9d as indicated by the arrows aftertreatment with Applicant's invention.

With respect to H. A., male, and FIG. 8, FIG. 8 indicates two areas ofbasal cell carcinoma by the arrows, close-ups of which are shown inFIGS. 8a and 8c. After treatment with the NSAIDS and HA gel three timesa day for the period between Jan. 26, 1992 and Mar. 16, 1992 the basalcell carcinoma is clearing as per FIGS. 8b and 8d.

The same is true with respect to male M. F. and FIG. 7 which appearsclear in the photographs (see FIG. 7a and the response shown in FIG.7b).

With respect to male, W. D. and FIG. 6, the upper lesion in FIG. 6a(indicated by the upper arrow) is gone after treatment with Applicant'sinvention (See FIG. 6b) and the two lower lesions in FIG. 6a are well ontheir way to disappearing (See FIG. 6b).

With respect to female D and FIG. 11, the lesion was left untreated fora long period and gradually encompassed her eye. Surgery could not beundertaken without jeopardizing the eye. By applying Applicant'sinvention (dosage amounts) over a prolonged period, the basal cellcarcinoma has constantly decreased in size.

With respect to FIG. 12, (12a) shows mice having tumors in the skininduced in their hind legs. After continuous applications to the shavedhind legs having the tumors in the skin by rubbing in dosage amounts byApplicant's invention, the tumors have decreased in size. (See FIG. 12b)

The effect of Hyaluronic acid as a drug carrier of anti-cancer agent(5-FU) 5-Fluoracil was also studied. (Intratumour injection study)

B. EXPERIMENTAL MODEL (2) 1. Method and Material

a. Animal: Fisher 344 rat, male 200-250 g

b. Tumor model Fisher Bladder Carcinoma Tumor (2 mm viable tumorfragment) was transplanted subcutaneously on the right frank by trocar

c. Treatment was started when tumor size is about 1.5 cm. (2 weeks afterimplantation.)

1. These drugs were administrated by intratumor injection. (right frank)

At the same time, injection into normal skin (left frank) was carriedout similarly.

    ______________________________________                                        Group A:  H-5-FU 5 mg/kg + saline/0.3 ml (i.t.)                               B:        H-5-FU 5 mg/kg + HA 15 mg/kg/0.3 ml (s.c.)                          ______________________________________                                    

3H-FU without or with HA was injected as a single dose (0.3 ml) into thecenter of the tumor (on the right frank) with a 30 gause needle. At thesame time, injection into normal skin (on the left frank) was carriedout similarly.

The tumor and skin was then removed at different times (1 h,6 hr) forcounting radioactivity of the remaining content in the tissue.

2. Result

All the results were expressed as Mean+S.E. under the followingheadings:

    ______________________________________                                        TUMOR TISSUE       NORMAL SKIN                                                (left hand portion (Right hand portion                                        of the graph)      of the graph)                                              ______________________________________                                         5-FU + HAgroup (n = 4)                                                                           5-FU + HAgroup (n = 4)                                     5-FU group (n = 4)                                                                               5-FU group (n = 4)                                        ______________________________________                                    

(See FIG. 4' of Page 4/12)

3. Conclusion

1. In 5-FU HA group radioactivity was accumulated and retained in thetumor tissue for a long period, whereas rapid clearance was demonstratedin normal tissue. (skin)

2. In 5-FU group, radioactivity immediately disappeared from the tumoror the normal tissue by diffusion, primarily into bloodcapillaries. - - - 5FU can traverse freely between the interstitialspace and blood capillary.

The Effect of Hyaluronic Acid as a Drug Carrier in Target CancerChemotherapy

A. EXPERIMENTAL MODEL (1) Intravenous Injection) 1. Method and Material

a. Animal: Fisher 344 rat, male 200-250 g

b. Tumor model Fisher Bladder Carcinoma Tumor (2 mm viable tumorfragment) was transplanted subcutaneously on the right frank by trocar

c. Treatment was started when tumor size is about 1.5 cm.(2 weeks afterimplantation.) . . . tumor weight:1.0±0.3 g The drug was administeredIntravenously (through the penile vein)

Group A: 5-FU 20 mg/kg (3H-5-FU30μCi)+saline B: 5-FU 20 mg/kg(3H-5-FU30μCi)+HA 15 mg/kg C: 5-FU 20 mg/kg (3H-5-FU30μCi)+HA 15mg/kg+(3H-HA30μCi)

2. Sample Collection

a. accumulation of ADR, 5-FU in tumor tissue and liver

(1). Tumor was surgically removed (and blood was collected) at*predeterminated time after drug administration. Tumor weight wasmeasured (and blood was centrifuged to obtain a plasma sample.) * 15min, 60 min, 3 hr, 4 hrs, . . . after drug administration . . . Liverwas removed for radioactivity counting at the same time.

(2). Radioactivity level in tumor tissue was counted, using a liquidscintillation counter.

    ______________________________________                                        3. Conclusion                                                                 Radioactivity in Tumor Tissue and Liver                                                         Tumor   Liver                                               ______________________________________                                        15 min                                                                              3H-5FU       (n = 6)  2810 ± 165                                                                         18680 ± 625                                  3H-5FU + HA  (n = 6)   352 ± 190                                                                         23593 ± 1460                                 3H-5FU + 3H-HA                                                                             (n = 4)  4087 ± 681                                                                         32060 ± 2145                           60 min                                                                              3H-5FU       (n = 3)  1751 ± 149                                                                         5451 ± 841                                   3H-5FU + HA  (n = 4)  2599 ± 489                                                                          8265 ± 1849                           3 hrs 3H-5FU       (n = 6)  1493 ± 227                                                                         2230 ± 449                                   3H-5FU + HA  (n = 6)  2512 ± 449                                                                         2897 ± 340                                   3H-5FU + 3H-HA                                                                             (n = 4)  3606 ± 929                                                                          6977 ± 1633                           5 hrs 3H-FU        (n = 3)   853 ± 129                                                                         1129 ± 70                                    3H-5FU + HA  (n = 3)  1981 ± 479                                                                         1754 ± 248                                   3H-5FU + 3H-HA                                                                             (n = 3)  2168 ± 163                                                                         3018 ± 325                             ______________________________________                                         mean ± S.E.                                                                HA: 15 mg/kg (30 μCi/kg)                                                   5FU: 20 mg/kg (30 μCi/kg)                                             

See FIG. 5' of Page 5/12 of the Figures which comprises a graph entitled"RADIOACTIVITY IN TUMOR TISSUE" comparing CPM on the vertical with timein Minutes on the horizontal (for example 100, 200, 300).

1. Radioactivity in tumor tissue in 5-FU+HA group is higher than that in5-FU group. There is significant difference (p>0.05, ANOVA) between withand without HA at 3 hrs after injection. The high intratumorconcentration was retained for a prolonged time in 5-FU+HA group. (Thisretention was confirmed by the intratumor injection study.)

2. These results teach that HA can enhance 5-FU uptake in tumor tissue.This phenomenon results from HA distribution (in tumor tissue HA may belost from the extracellular matrix) and the vascular uniqueness of tumortissue (hyperpermiability of tumor vessels to macromolecular drug, HA).

As many changes can be made to the invention without departing from thescope of the invention, it is intended that all material containedherein be interpreted as illustrative of the invention and not in alimiting sense.

The embodiments of the invention in which an exclusive property orprivilege is claimed are as follows:
 1. A pharmaceutical compositionfrom which an effective non-toxic dosage amounts are to be taken andapplied topically to the skin or exposed tissue of a human, eacheffective dosage amount consisting essentially of pharmaceuticalexcipients suitable for topical application, an effective non-toxicdosage amount of a drug to treat diseases and conditions of the skin orexposed tissue of a human and an effective non-toxic dosage amount of aform of hyaluronic acid selected from the group consisting of hyaluronicacid, non-toxic salts thereof and combination thereof having a molecularweight less than 750,000 daltons and greater than 150,000 daltonssufficient to transport the drug percutaneously into the epidermis ofthe skin or exposed tissue of a site in need of treatment wherein thecomposition accumulates and remains in the epidermis for a prolongedperiod of time and which is systemic independent acting and wherein thedrug in the pharmaceutical composition is between about 1% and about 5%by weight of the composition and the amount of the form of hyaluronicacid in the composition is between about 1% and about 3% by weight ofthe composition and wherein each dosage to be taken comprises a minimumof 5 mg of the form of hyaluronic acid per square centimeter of skin orexposed tissue to which it is to be applied.
 2. The composition of claim1 wherein the drug is a non-steroidal anti-inflammatory agent (NSAID) inan amount between about 1% and about 5% of the composition by weight andthe pharmaceutical excipients include a preservative and a solubilizerfor the NSAID.
 3. The composition of claim 2 wherein the form of thecomposition is selected from the group consisting of a gel and creamsuitable for topical application and wherein the NSAID is 3% by weightdiclofenac, the form of hyaluronic acid is present as 21/2% by weight ofthe composition, and the pharmaceutical excipients include a solubilizerfor solubilizing the diclofenac, and a preservative.
 4. The compositionof claim 3 wherein the preservative is benzyl alcohol present as 1% byweight of the composition and the solubilizer is methoxypolyethyleneglycol present as 20% by weight of the composition.
 5. The compositionof claim 3 suitable for topical application wherein the pharmaceuticalexcipients include 20% by weight methoxypolyethylene glycol, 1% byweight benzyl alcohol, 3% by weight diclofenac sodium, 2.5% by weightsodium hyaluronate, and the balance is sterile water.
 6. The compositionof claim 2 wherein the form of said composition is in a form for topicalapplication, further includingbetween about 1 to about 3% by weight ofthe composition of sodium hyaluronate having a molecular weight lessthan about 750,000 daltons and greater than about 150,000 daltonsbetween about 1 to about 5% by weight of the composition of an NSAID andthe balance selected from the group consisting of excipients suitablefor topical application and water.
 7. A dosage amount of apharmaceutical composition consisting essentially of(1) pharmaceuticalexcipients suitable for topical application including water; (2) atherapeutic agent in a therapeutically effective amount to treat adisease or condition of the skin and exposed tissue and; (3) a form ofhyaluronic acid selected from the group consisting of hyaluronic acidand non-toxic salts thereof, having a molecular weight less than 750,000daltons and greater than 150,000 daltons, characterized in that saiddosage amount of said composition is in a dosage form suitable fortopical application to the skin and exposed tissue, is in a dosageamount in which component (2) exceeds 5 mg/cm² of the skin or exposedtissue to which the dosage amount is to be applied, and is in such formthat component (3) is immediately available to transport component (2)percutaneously into the epidermis of the skin or exposed tissue to thesite of trauma and pathology of the disease or condition to be treatedin the skin or exposed tissue, where the dosage amount of thecomposition accumulates (in the epidermis) for a prolonged period beforepassage therefrom, and wherein the concentrations by weight ofcomponents (2) and (3) in the dosage amount are selected from:(i)component (3) equals or is less than 3% by weight of the dosage amountbut equal to or greater than 1% by weight of the dosage amount andcomponent (2) equals or is less than 5% by weight of the dosage amountbut equal to or greater than 1% by weight of the dosage amount, and (ii)component (3) is about 21/2% by weight of the dosage amount andcomponent (2) is 3% by weight of the dosage amount.
 8. The dosage amountof claim 7 wherein the concentration of components (2) and (3) in thedosage amounts is that in subparagraph (i).
 9. The dosage amount ofclaim 7 wherein the concentration of components (2) and (3) in thedosage amounts is that in subparagraph (ii).
 10. The dosage amount ofclaim 8 wherein component (2) is an NSAID.
 11. The dosage amount ofclaim 9 wherein component (2) is an NSAID.
 12. The dosage amount ofclaim 10 wherein component (3) exceeds 10 mg/cm².
 13. The dosage amountof claim 11 wherein component (3) exceeds 10 mg/cm².